Immunosuppressive regimens and methods thereof

Abstract

In particular, the Disclosure provides methods, for example, methods for treating a subject, methods for immunosuppression, comprising administering recombinant adeno-associated virus vectors (rAAVs) and immunosuppressive regimens. In some embodiments, the Disclosure provides methods for reducing dorsal root ganglion (DRG) toxicity. In some embodiments, the Disclosure provides immunosuppressive regimens comprising, for example, dexamethasone and one or more immunosuppressants, for example, calcineurin inhibitors. In some aspects, the Disclosure provides methods for treating a subject, comprising administering recombinant adeno-associated virus vectors (rAAVs) and administering immunosuppressive regimens.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 459,413, filed on 14 April 2023, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Gene therapy using adeno-associated virus vectors (rAAVs) is an important and developing modality for treating numerous diseases, disorders, and conditions. The use of rAAVs is characterized by several challenges, including toxicity that can affect specific organs, tissues, or cells in recipients. For example, dorsal root ganglion toxicity (e.g., lesions) associated with the administration of rAAVs, particularly those targeting the central nervous system (CNS), remains an ongoing challenge. Such toxicity can result from an immune response to one or more components of the rAAV. Therefore, therapeutic techniques to reduce or mitigate rAAV-related toxicity remain necessary. [Overview of the project] [Means for solving the problem]

[0003] In some embodiments, the Disclosure provides a method for treating a subject, comprising administering a recombinant adeno-associated virus vector (rAAV) and administering an immunosuppressive regimen.

[0004] In some embodiments, the immunosuppressive regimen comprises dexamethasone and a calcineurin inhibitor. In some embodiments, the calcineurin inhibitor comprises a macrolide. In some embodiments, the calcineurin inhibitor comprises tacrolimus.

[0005] In some embodiments, rAAV includes a nucleic acid sequence encoding a polypeptide. In some embodiments, rAAV includes a nucleic acid sequence encoding an RNA molecule. In some embodiments, rAAV includes a nucleic acid sequence encoding miRNA. In some embodiments, rAAV includes a nucleic acid sequence encoding a polypeptide and a nucleic acid sequence encoding an RNA molecule.

[0006] In some embodiments, rAAV is administered intravenously, intrathecally, intraventricularly, intracisionally, intramuscularly, intraparenchymally, intracranially, intraocularly, intraarticularly, intranasally, and / or subcutaneously. In some embodiments, rAAV is administered intravenously.

[0007] In some embodiments, the immunosuppressive regimen comprises dexamethasone and a calcineurin inhibitor, as well as one or more additional immunosuppressants. In some embodiments, the immunosuppressive regimen comprises dexamethasone and a calcineurin inhibitor, as well as two or more additional immunosuppressants. In some embodiments, the immunosuppressive regimen comprises dexamethasone and a calcineurin inhibitor, as well as three or more additional immunosuppressants. In some embodiments, the immunosuppressive regimen comprises dexamethasone and a calcineurin inhibitor, as well as inosine monophosphate dehydrogenase (IMPDH) and / or Janus kinase (JAK) inhibitors. In some embodiments, the immunosuppressive regimen comprises dexamethasone, a calcineurin inhibitor, and an IMPDH inhibitor. In some embodiments, the immunosuppressive regimen comprises dexamethasone, a calcineurin inhibitor, and a JAK inhibitor. In some embodiments, the immunosuppressive regimen comprises dexamethasone, a calcineurin inhibitor, an IMPDH inhibitor, and a JAK inhibitor. In some embodiments, the IMPDH inhibitor comprises mycophenolate mofetil (MMF). In some embodiments, the JAK inhibitor comprises tofacitinib.

[0008] In some embodiments, dexamethasone is administered intraosseously, intrathecally, intravenously, and / or orally. In some embodiments, dexamethasone is administered intraosseously. In some embodiments, dexamethasone is administered intrathecally. In some embodiments, dexamethasone is administered intravenously. In some embodiments, dexamethasone is administered in doses of approximately 0.01 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 5 mg / kg, approximately 0.25 mg / kg to approximately 2.5 mg / kg, or approximately 0.5 mg / kg to approximately 1.0 mg / kg. In some embodiments, dexamethasone is administered orally. In some embodiments, dexamethasone is administered daily.

[0009] In some embodiments, dexamethasone is administered (i) on each of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days prior to the administration of rAAV, (ii) on the same day as the administration of rAAV, and / or (iii) on each day after the administration of rAAV, for a period of approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer.

[0010] In some embodiments, the calcineurin inhibitor is administered orally and / or intravenously. In some embodiments, the calcineurin inhibitor is administered orally. In some embodiments, the calcineurin inhibitor is administered intravenously. In some embodiments, the calcineurin inhibitor is administered in doses of approximately 0.01 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 5 mg / kg, approximately 0.25 mg / kg to approximately 2.5 mg / kg, or approximately 0.5 mg / kg to approximately 1 mg / kg. In some embodiments, the calcineurin inhibitor is administered daily.

[0011] In some embodiments, the calcineurin inhibitor is administered for approximately one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or longer, on each of the following days (i) on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, or more days prior to the administration of rAAV, (ii) on the same day as the administration of rAAV, and / or (iii) on each day after the administration of rAAV.

[0012] In some embodiments, MMF is administered orally or intravenously. In some embodiments, MMF is administered orally. In some embodiments, MMF is administered intravenously. In some embodiments, MMF is administered in doses of approximately 0.1 mg / kg to approximately 200 mg / kg, approximately 1 mg / kg to approximately 100 mg / kg, approximately 10 mg / kg to approximately 75 mg / kg, or approximately 25 mg / kg to approximately 50 mg / kg. In some embodiments, MMF is administered daily. In some embodiments, MMF is administered twice daily.

[0013] In some embodiments, MMF is administered (i) on each of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days prior to the administration of rAAV, (ii) on the same day as the administration of rAAV, and / or (iii) on each day after the administration of rAAV, for a period of approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer.

[0014] In some embodiments, tofacitinib is administered orally or intravenously. In some embodiments, tofacitinib is administered orally. In some embodiments, tofacitinib is administered intravenously. In some embodiments, tofacitinib is administered in doses of approximately 0.01 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 5 mg / kg, approximately 0.25 mg / kg to approximately 2.5 mg / kg, or approximately 0.5 mg / kg to approximately 1 mg / kg. In some embodiments, tofacitinib is administered daily.

[0015] In some embodiments, tofacitinib is administered for approximately one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or longer, on each of the following days (i) on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, or more days prior to the administration of rAAV, (ii) on the same day as the administration of rAAV, and / or (iii) on each day after the administration of rAAV.

[0016] In some embodiments, the subject is a mammal. In some embodiments, the subject is a mouse. In some embodiments, the subject is a rat. In some embodiments, the subject is a rabbit. In some embodiments, the subject is a dog. In some embodiments, the subject is a pig. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a human. In some embodiments, the subject is an adult. In some embodiments, the subject is a child.

[0017] In some embodiments, dorsal root ganglion (DRG) toxicity is reduced compared to rAAV administration without an immunosuppressive regimen. In some embodiments, the severity and / or incidence of lesions in one or more DRGs is reduced compared to rAAV administration without an immunosuppressive regimen. In some embodiments, the severity of lesions in one or more DRGs is reduced compared to rAAV administration without an immunosuppressive regimen. In some embodiments, the incidence of lesions in one or more DRGs is reduced compared to rAAV administration without an immunosuppressive regimen. In some embodiments, axonal degeneration in one or more DRGs is reduced compared to rAAV administration without an immunosuppressive regimen. [Brief explanation of the drawing]

[0018] [Figure 1A]Immune cell foci are present in the dorsal root ganglia (DRG) of animals administered rAAV. Cynomolgus monkeys were administered 3.68 × 10^13 genome copies (GC) of rAAV containing a nucleic acid sequence encoding a polypeptide via single intracisternal (ICM) injection. The control group of animals received only vehicle (artificial cerebrospinal fluid; aCSF). At 5, 9, 15, and 29 days after rAAV administration, the animals were sacrificed for histopathological analysis of the DRG. Immunohistochemistry was performed using a monoclonal anti-CD68 antibody (KP1) to identify CD68+ macrophage infiltration. Representative images at each time point are shown. [Figure 1B] Immune cell foci are present in the dorsal root ganglia (DRG) of animals administered rAAV. Cynomolgus monkeys were administered 3.68 × 10^13 genome copies (GC) of rAAV containing a nucleic acid sequence encoding a polypeptide via single intracisternal (ICM) injection. The control group of animals received only vehicle (artificial cerebrospinal fluid; aCSF). At 5, 9, 15, and 29 days after rAAV administration, the animals were sacrificed for histopathological analysis of the DRG. Immunohistochemistry was performed using a monoclonal anti-CD68 antibody (KP1) to identify CD68+ macrophage infiltration. The CD68+ area was quantified using Visiopharm image analysis software. Each column (e.g., 1001, 1002, 1501) represents one animal. The size of each plotted circle represents the CD68+ % area. The Y-axis of the graph indicates the spinal cord level.

[0019] [Figure 2A] Immune cell foci are present in the DRG of animals administered rAAV. Cynomolgus monkeys were administered 3.68 × 10^13 GC of rAAV containing a nucleic acid sequence encoding a polypeptide via single ICM injection. The control group of animals received only vehicle (artificial cerebrospinal fluid; aCSF). At 5, 9, 15, and 29 days after rAAV administration, the animals were sacrificed for histopathological analysis of the DRG. Immunohistochemistry was performed using a monoclonal anti-CD4 antibody (EPR6855) to identify CD4+ T cell infiltration. Representative images at each time point are shown. [Figure 2B]Immune cell lesions were present in the DRG of animals administered rAAV. Cynomolgus monkeys were administered 3.68 × 10¹³ GC of rAAV containing a polypeptide-encoding nucleic acid sequence via a single ICM injection. The animal control group received only the vehicle (artificial cerebrospinal fluid; aCSF). Animals were sacrificed for histopathological analysis of the DRG on days 5, 9, 15, and 29 after rAAV administration. Immunohistochemistry using monoclonal anti-CD4 antibody (EPR6855) was performed to identify CD4+ T cell infiltration. CD4+ area was quantified using Visiopharm image analysis software. Each column (e.g., 1001, 1002, 1501) represents one animal. The size of each plotted circle represents the CD4+ % area. The Y-axis of the graph represents the spinal cord level.

[0020] [Figure 3A] Immune cell lesions are present in the DRG of animals administered with rAAV. Cynomolgus monkeys were administered 3.68 × 10¹³ GC of rAAV containing a polypeptide-encoding nucleic acid sequence via a single ICM injection. The animal control group received only the vehicle (artificial cerebrospinal fluid; aCSF). Animals were sacrificed for histopathological analysis of the DRG at days 5, 9, 15, and 29 after rAAV administration. Immunohistochemistry using monoclonal anti-CD20 antibody (L26) was performed to identify CD20+ B cell infiltration. Representative images at each time point are shown. [Figure 3B]Immune cell lesions were present in the DRG of animals administered rAAV. Cynomolgus monkeys were administered 3.68 × 10¹³ GC of rAAV containing a polypeptide-encoding nucleic acid sequence via a single ICM injection. The animal control group received only the vehicle (artificial cerebrospinal fluid; aCSF). Animals were sacrificed for histopathological analysis of the DRG on days 5, 9, 15, and 29 after rAAV administration. Immunohistochemistry using monoclonal anti-CD20 antibody (L26) was performed to identify CD20+ B cell infiltration. CD20+ area was quantified using Visiopharm image analysis software. Each column (e.g., 1001, 1002, 1501) represents one animal. The size of each plotted circle represents the CD20+% area. The Y-axis of the graph represents the spinal cord level.

[0021] [Figure 4A] Immune cell lesions are present in the DRG of animals administered with rAAV. Cynomolgus monkeys were administered 3.68 × 10¹³ GC of rAAV containing a polypeptide-encoding nucleic acid sequence via a single ICM injection. The animal control group received only the vehicle (artificial cerebrospinal fluid; aCSF). Animals were sacrificed for histopathological analysis of the DRG at days 5, 9, 15, and 29 after rAAV administration. Immunohistochemistry using monoclonal anti-CD8 antibody (SP57) was performed to identify CD8+ T cell infiltration. Representative images at each time point are shown. [Figure 4B] Immune cell lesions were present in the DRG of animals administered rAAV. Cynomolgus monkeys were administered 3.68 × 10¹³ GC of rAAV containing a polypeptide-encoding nucleic acid sequence via a single ICM injection. The animal control group received only the vehicle (artificial cerebrospinal fluid; aCSF). Animals were sacrificed for histopathological analysis of the DRG on days 5, 9, 15, and 29 after rAAV administration. Immunohistochemistry using monoclonal anti-CD8 antibody (SP57) was performed to identify CD8+ T cell infiltration. CD8+ area was quantified using Visiopharm image analysis software. Each column (e.g., 1001, 1002, 1501) represents one animal. The size of each plotted circle represents the CD8+ % area. The Y-axis of the graph represents the spinal cord level.

[0022] [Figure 5A] Immune cell lesions are present in the DRG of animals administered rAAV. Cynomolgus monkeys were administered 3.68 × 10¹³ GC of rAAV containing a polypeptide-encoding nucleic acid sequence via a single ICM injection. The animal control group received only the vehicle (artificial cerebrospinal fluid; aCSF). Animals were sacrificed for histopathological analysis of the DRG at days 5, 9, 15, and 29 after rAAV administration. NK cell infiltration was identified by in-situ hybridization using probes to detect NCR1 / Nkp46 mRNA. A negative control probe for dapB (bacterial gene 1) was also used to confirm the specificity of the reaction. Representative images at each time point are shown. Arrows point to regions of NCR1 / Nkp46+ signaling. [Figure 5B] Immune cell lesions were present in the DRG of animals administered rAAV. Cynomolgus monkeys were administered 3.68 × 10¹³ GC of rAAV containing a polypeptide-encoding nucleic acid sequence via a single ICM injection. The animal control group received only the vehicle (artificial cerebrospinal fluid; aCSF). Animals were sacrificed for histopathological analysis of the DRG on days 5, 9, 15, and 29 after rAAV administration. NK cell infiltration was identified by in-situ hybridization using probes to detect NCR1 / Nkp46 mRNA. A negative control probe for dapB (bacterial gene 1) was also used to confirm the specificity of the reaction. NCR1 / Nkp46+ area was quantified using Visiopharm image analysis software. Each column (e.g., 1001, 1002, 1501) represents one animal. The size of each plotted circle represents the NCR1 / Nkp46+% area. The Y-axis of the graph represents the spinal cord level.

[0023] [Figure 6]The technology provided can reduce dorsal root ganglion, trigeminal ganglion, and motor neuron toxicity. Cynomolgus monkeys were administered 3 × 10¹³ GC of rAAV containing a polypeptide-encoding nucleic acid sequence via a single ICM injection, either alone or in combination with an immunosuppressive regimen (IS) including dexamethasone (intrathecal, once; orally, daily), tacrolimus (oral, daily), and mycophenolate mofetil (MMF) (oral, twice daily). A control group of animals received neither rAAV nor an immunosuppressive regimen. Four weeks after rAAV administration, the animals were sacrificed for histopathological analysis of lesions in the dorsal root ganglion (DRG), spinal cord (SC), and trigeminal ganglion (TG). Each column (e.g., 1001, 1002, 1501) represents one animal. For the top five rows, the three bar columns represent tissue collected from the lumbar, thoracic, and cervical levels, respectively, from left to right. The Y-axis of each graph represents the severity score (0 = no lesion (0% of tissue affected); 1 = minimal (<10% of tissue affected); 2 = mild (10-25% of tissue affected); 3 = moderate (25-50%); 4 = significant (50-95%); 5 = severe (>95%)).

[0024] [Figure 7-1]The technology provided can reduce dorsal root ganglion, spinal cord, and sympathetic ganglion toxicity. Cynomolgus monkeys were administered rAAV in 1 × 10¹³, 3 × 10¹³, or 1 × 10¹⁴ GC, containing nucleic acid sequences encoding polypeptides, either alone or in combination with an immunosuppressive regimen (IS) including dexamethasone (oral, daily), tacrolimus (oral, daily), and MMF (oral, daily). The animal control group received only a vehicle (artificial cerebrospinal fluid; aCSF) control. Each column (e.g., 1001, 1002, 1501) represents one animal. Each row in the graph represents the scored lesion severity for the region (DRG = dorsal root ganglion; SC = spinal cord; SG = sympathetic ganglion) and lesion type (e.g., neurodegeneration, mononuclear cell infiltration) listed on the left. The Y-axis of each graph represents the spinal cord region (cervical, thoracic, lumbar, sacral). The size of the circle represents the severity score of the lesion (no circle = no lesion (0% of tissue affected), 1 = minimal (<10% of tissue affected), 2 = mild (10-25% of tissue affected), 3 = moderate (25-50%), 4 = significant (50-95%), 5 = severe (>95%)). [Figure 7-2]The technology provided can reduce dorsal root ganglion, spinal cord, and sympathetic ganglion toxicity. Cynomolgus monkeys were administered rAAV in 1 × 10¹³, 3 × 10¹³, or 1 × 10¹⁴ GC, containing nucleic acid sequences encoding polypeptides, either alone or in combination with an immunosuppressive regimen (IS) including dexamethasone (oral, daily), tacrolimus (oral, daily), and MMF (oral, daily). The animal control group received only a vehicle (artificial cerebrospinal fluid; aCSF) control. Each column (e.g., 1001, 1002, 1501) represents one animal. Each row in the graph represents the scored lesion severity for the region (DRG = dorsal root ganglion; SC = spinal cord; SG = sympathetic ganglion) and lesion type (e.g., neurodegeneration, mononuclear cell infiltration) listed on the left. The Y-axis of each graph represents the spinal cord region (cervical, thoracic, lumbar, sacral). The size of the circle represents the severity score of the lesion (no circle = no lesion (0% of tissue affected), 1 = minimal (<10% of tissue affected), 2 = mild (10-25% of tissue affected), 3 = moderate (25-50%), 4 = significant (50-95%), 5 = severe (>95%)).

[0025] [Figure 8-1]The technology provided can reduce dorsal root ganglia, nerve roots, spinal cord, and peripheral neurotoxicity. Cynomolgus monkeys were administered 2×10¹³, 4×10¹³, or 7.5×10¹³ GC of rAAV containing a miRNA-encoding nucleic acid sequence via a single intrathecal (IT) injection, either alone or in combination with immunosuppressive regimens including dexamethasone, tacrolimus, and tofacitinib ("Triple"), dexamethasone and tacrolimus ("Dex+Tacro"), or dexamethasone and tofacitinib ("Dex+Tofa"). Dexamethasone and tacrolimus were administered orally daily, and tofacitinib was administered orally twice daily. The animal control group received only vehicle controls. Each column (e.g., 1001, 1501, 1502) represents one animal. The first five rows of the graphs represent the scored lesion severity for the region (DRG = dorsal root ganglion) and lesion type (e.g., neurodegeneration, mononuclear cell infiltration) listed on the left. The Y-axis of each graph represents the spinal cord level. The size of the circles represents the lesion severity score (no circle = no lesion (0% of tissue affected), 1 = minimal (<10% of tissue affected), 2 = mild (10-25% of tissue affected), 3 = moderate (25-50%), 4 = significant (50-95%), 5 = severe (>95%)). The graph in the last row depicts the measured levels of phosphorylated nerve filament heavy (pNfH) chains in cerebrospinal fluid (CSF). The Y-axis of the graphs represents pg*day / ml. [Figure 8-2]The technology provided can reduce dorsal root ganglia, nerve roots, spinal cord, and peripheral neurotoxicity. Cynomolgus monkeys were administered 2×10¹³, 4×10¹³, or 7.5×10¹³ GC of rAAV containing a miRNA-encoding nucleic acid sequence via a single intrathecal (IT) injection, either alone or in combination with immunosuppressive regimens including dexamethasone, tacrolimus, and tofacitinib ("Triple"), dexamethasone and tacrolimus ("Dex+Tacro"), or dexamethasone and tofacitinib ("Dex+Tofa"). Dexamethasone and tacrolimus were administered orally daily, and tofacitinib was administered orally twice daily. The animal control group received only vehicle controls. Each column (e.g., 1001, 1501, 1502) represents one animal. The first five rows of the graphs represent the scored lesion severity for the region (DRG = dorsal root ganglion) and lesion type (e.g., neurodegeneration, mononuclear cell infiltration) listed on the left. The Y-axis of each graph represents the spinal cord level. The size of the circles represents the lesion severity score (no circle = no lesion (0% of tissue affected), 1 = minimal (<10% of tissue affected), 2 = mild (10-25% of tissue affected), 3 = moderate (25-50%), 4 = significant (50-95%), 5 = severe (>95%)). The graph in the last row depicts the measured levels of phosphorylated nerve filament heavy (pNfH) chains in cerebrospinal fluid (CSF). The Y-axis of the graphs represents pg*day / ml. [Figure 8-3]The technology provided can reduce dorsal root ganglia, nerve roots, spinal cord, and peripheral neurotoxicity. Cynomolgus monkeys were administered 2×10¹³, 4×10¹³, or 7.5×10¹³ GC of rAAV containing a miRNA-encoding nucleic acid sequence via a single intrathecal (IT) injection, either alone or in combination with immunosuppressive regimens including dexamethasone, tacrolimus, and tofacitinib ("Triple"), dexamethasone and tacrolimus ("Dex+Tacro"), or dexamethasone and tofacitinib ("Dex+Tofa"). Dexamethasone and tacrolimus were administered orally daily, and tofacitinib was administered orally twice daily. The animal control group received only vehicle controls. Each column (e.g., 1001, 1501, 1502) represents one animal. The first five rows of the graphs represent the scored lesion severity for the region (DRG = dorsal root ganglion) and lesion type (e.g., neurodegeneration, mononuclear cell infiltration) listed on the left. The Y-axis of each graph represents the spinal cord level. The size of the circles represents the lesion severity score (no circle = no lesion (0% of tissue affected), 1 = minimal (<10% of tissue affected), 2 = mild (10-25% of tissue affected), 3 = moderate (25-50%), 4 = significant (50-95%), 5 = severe (>95%)). The graph in the last row depicts the measured levels of phosphorylated nerve filament heavy (pNfH) chains in cerebrospinal fluid (CSF). The Y-axis of the graphs represents pg*day / ml.

[0026] [Figure 9]Exemplary peripheral drug exposure in blood and plasma. Cynomolgus monkeys were administered 5 × 10¹³ vg / kg of rAAV containing a nucleic acid sequence encoding the mCherry protein via a single intravenous (IV) injection in combination with an immunosuppressive regimen containing dexamethasone and tacrolimus (Dex / Tac) (N=3) or an immunosuppressive regimen containing prednisolone (Pred) (N=3). A control group of animals (N=2) received a vehicle control in combination with dexamethasone and tacrolimus. Dexamethasone, tacrolimus, and / or prednisolone were administered orally once daily. Blood and plasma samples were collected from each animal on day 21, 4 hours after administration of the immunosuppressive regimen. The X-axis of the graph represents the immunosuppressant (dexamethasone, tacrolimus, or prednisolone), and the Y-axis depicts the ng / ml of the immunosuppressant detected in the blood for dexamethasone and prednisolone, or in the plasma for tacrolimus. Each circle represents data collected from one animal.

[0027] [Figure 10A]The technology provided can offer in vivo reduction of nerve damage. Cynomolgus monkeys were administered 5 × 10¹³ vg / kg of rAAV containing the nucleic acid sequence encoding the mCherry protein via a single intravenous (IV) injection in combination with an immunosuppressive regimen containing dexamethasone and tacrolimus (Dex / Tac) (N=3) or an immunosuppressive regimen containing prednisolone (Pred) (N=3). A control group of animals (N=2) received a vehicle control in combination with dexamethasone and tacrolimus. Dexamethasone, tacrolimus, and / or prednisolone were administered orally once daily. Blood samples were collected from each animal on day 21, 4 hours after administration with the immunosuppressive regimen, and the levels of nerve filament subunit NF-H in the plasma were examined. NF-H was quantified using enzyme-linked immunosorbent assay (ELISA) (Bio-Techne) as a biomarker of nerve damage. The columns represent the mean values ​​for each treatment group. The shape (square, circle, triangle) indicates the measurements for individual animals within each treatment group. Animal identifiers (e.g., 2001, 2501, 2002) are shown in the legend on the right. The X-axis of the graph represents the treatment group, and the Y-axis of the graph depicts the picograms (pg) of detected NF-H per milliliter (mL). [Figure 10B]The technology provided can offer in vivo reduction of nerve damage. Cynomolgus monkeys were administered 5 × 10¹³ vg / kg of rAAV containing the nucleic acid sequence encoding the mCherry protein via a single intravenous (IV) injection in combination with an immunosuppressive regimen containing dexamethasone and tacrolimus (Dex / Tac) (N=3) or an immunosuppressive regimen containing prednisolone (Pred) (N=3). A control group of animals (N=2) received a vehicle control in combination with dexamethasone and tacrolimus. Dexamethasone, tacrolimus, and / or prednisolone were administered orally once daily. Blood samples were collected from each animal on day 21, 4 hours after administration with the immunosuppressive regimen, and the levels of nerve filament subunit NF-H in the plasma were examined. Representative images of mCherry protein expression (dark stained areas) are shown. The treatment group is shown on the left. The insertion numbers (2001, 2501, 2002, 5001, 5502, 5601) indicate the animals from which the depicted dorsal root ganglion (DRG) tissue was collected. The magnification is shown at the top. The rightmost column of the 40x magnification images is an enlargement of the area from the 10x magnification images of 2001 (top) and 5601 (bottom). [Figure 10C-1]The technology provided can offer in vivo reduction of nerve damage. Cynomolgus monkeys were administered 5 × 10¹³ vg / kg of rAAV containing the nucleic acid sequence encoding the mCherry protein via a single intravenous (IV) injection in combination with an immunosuppressive regimen containing dexamethasone and tacrolimus (Dex / Tac) (N=3) or an immunosuppressive regimen containing prednisolone (Pred) (N=3). A control group of animals (N=2) received a vehicle control in combination with dexamethasone and tacrolimus. Dexamethasone, tacrolimus, and / or prednisolone were administered orally once daily. Blood samples were collected from each animal on day 21, 4 hours after administration with the immunosuppressive regimen, and levels of nerve filament subunit NF-H in the plasma were examined. At the end of the study, the animals were sacrificed, tissues were collected, and lesions were examined. The treatment groups of the animals are shown above. The size and fill of the circles represent the lesion severity score. For example, for animal 5001, infiltration in the dorsal root ganglia and sacrum, mononuclear cells were observed as mild; degeneration / necrosis in the dorsal root ganglia and sacrum, neurons were observed as minimal; and degeneration in the sciatic nerve, nerve fibers were not observed. [Figure 10C-2]The technology provided can offer in vivo reduction of nerve damage. Cynomolgus monkeys were administered 5 × 10¹³ vg / kg of rAAV containing the nucleic acid sequence encoding the mCherry protein via a single intravenous (IV) injection in combination with an immunosuppressive regimen containing dexamethasone and tacrolimus (Dex / Tac) (N=3) or an immunosuppressive regimen containing prednisolone (Pred) (N=3). A control group of animals (N=2) received a vehicle control in combination with dexamethasone and tacrolimus. Dexamethasone, tacrolimus, and / or prednisolone were administered orally once daily. Blood samples were collected from each animal on day 21, 4 hours after administration with the immunosuppressive regimen, and levels of nerve filament subunit NF-H in the plasma were examined. At the end of the study, the animals were sacrificed, tissues were collected, and lesions were examined. The treatment groups of the animals are shown above. The size and fill of the circles represent the lesion severity score. For example, for animal 5001, infiltration in the dorsal root ganglia and sacrum, mononuclear cells were observed as mild; degeneration / necrosis in the dorsal root ganglia and sacrum, neurons were observed as minimal; and degeneration in the sciatic nerve, nerve fibers were not observed.

[0028] [Figure 11A]The technology provided can reduce the increase in liver enzymes during AAV treatment. Cynomolgus monkeys were administered 5 × 10¹³ vg / kg of rAAV containing the nucleic acid sequence encoding the mCherry protein via a single intravenous (IV) injection in combination with an immunosuppressive regimen (N=3) containing dexamethasone and tacrolimus (Dex / Tac) or an immunosuppressive regimen (N=3) containing prednisolone (Pred). A control group of animals (N=2) received a vehicle control in combination with dexamethasone and tacrolimus. Dexamethasone, tacrolimus, and / or prednisolone were administered orally once daily. Blood samples were collected from the animals on days 7, 4, 8, 15, and 22 (0 = rAAV administration day) and subsequently analyzed for levels of certain liver enzymes and total bilirubin. Aspartate transaminase (AST) levels were quantified. The X-axis depicts the study day (0 = rAAV administration day), and the Y-axis depicts the detected AST units (U) per liter (L). Each graphed line corresponds to data collected from one animal. The legend on the right identifies the treatment group. From top to bottom on study day 15: The top three lines correspond to animals treated with rAAV + prednisolone, the next three lines correspond to animals treated with rAAV + dexamethasone / tacrolimus, and the bottom two lines correspond to animals treated with vehicle + dexamethasone / tacrolimus. [Figure 11B]The technology provided can reduce the increase in liver enzymes during AAV treatment. Cynomolgus monkeys were administered 5 × 10¹³ vg / kg of rAAV containing the nucleic acid sequence encoding the mCherry protein via a single intravenous (IV) injection in combination with an immunosuppressive regimen (N=3) containing dexamethasone and tacrolimus (Dex / Tac) or an immunosuppressive regimen (N=3) containing prednisolone (Pred). A control group of animals (N=2) received a vehicle control in combination with dexamethasone and tacrolimus. Dexamethasone, tacrolimus, and / or prednisolone were administered orally once daily. Blood samples were collected from the animals on days 7, 4, 8, 15, and 22 (0 = rAAV administration day) and subsequently analyzed for levels of certain liver enzymes and total bilirubin. Alanine transaminase (ALT) levels were quantified. The X-axis depicts the study day (0 = rAAV administration day), and the Y-axis depicts the units (U) of ALT detected per liter (L). Each graphed line corresponds to data collected from one animal. The legend on the right identifies the treatment group. From top to bottom on study day 22: The top three lines correspond to animals treated with rAAV + prednisolone, the next three lines correspond to animals treated with rAAV + dexamethasone / tacrolimus, and the bottom two lines correspond to animals treated with vehicle + dexamethasone / tacrolimus. [Figure 11C]The technology provided can reduce the increase in liver enzymes during AAV treatment. Cynomolgus monkeys were administered 5 × 10¹³ vg / kg of rAAV containing the nucleic acid sequence encoding the mCherry protein via a single intravenous (IV) injection in combination with an immunosuppressive regimen (N=3) containing dexamethasone and tacrolimus (Dex / Tac) or an immunosuppressive regimen (N=3) containing prednisolone (Pred). A control group of animals (N=2) received a vehicle control in combination with dexamethasone and tacrolimus. Dexamethasone, tacrolimus, and / or prednisolone were administered orally once daily. Blood samples were collected from the animals on days 7, 4, 8, 15, and 22 (0 = rAAV administration day) and subsequently analyzed for levels of certain liver enzymes and total bilirubin. Gamma-glutamyltransferase (GGT) levels were quantified. The X-axis depicts the study day (0 = rAAV administration day), and the Y-axis depicts the units (U) of detected GGT per liter (L). Each graphed line corresponds to data collected from one animal. The legend on the right identifies the treatment group. From top to bottom on study day = 15: The top three lines correspond to animals treated with rAAV + prednisolone, the next line corresponds to animals treated with rAAV + dexamethasone / tacrolimus, the next line corresponds to animals treated with vehicle + dexamethasone / tacrolimus, the next line corresponds to animals treated with vehicle + dexamethasone / tacrolimus, and the bottom line corresponds to animals treated with rAAV + dexamethasone / tacrolimus. [Figure 11D]The technology provided can reduce the increase in liver enzymes during AAV treatment. Cynomolgus monkeys were administered 5 × 10¹³ vg / kg of rAAV containing the nucleic acid sequence encoding the mCherry protein via a single intravenous (IV) injection in combination with an immunosuppressive regimen (N=3) containing dexamethasone and tacrolimus (Dex / Tac) or an immunosuppressive regimen (N=3) containing prednisolone (Pred). A control group of animals (N=2) received a vehicle control in combination with dexamethasone and tacrolimus. Dexamethasone, tacrolimus, and / or prednisolone were administered orally once daily. Blood samples were collected from the animals on days 7, 4, 8, 15, and 22 (0 = rAAV administration day) and subsequently analyzed for levels of certain liver enzymes and total bilirubin. Total bilirubin (TBIL) levels were quantified. The X-axis depicts the study day (0 = rAAV administration day), and the Y-axis depicts milligrams (mg) of total bilirubin per deciliter (dL). Each graphed line corresponds to data collected from one animal. The legend on the right identifies the treatment group. From top to bottom on study day = 15: The top three lines correspond to animals treated with rAAV + prednisolone, the next line corresponds to animals treated with vehicle + dexamethasone / tacrolimus, the next three lines correspond to animals treated with rAAV + dexamethasone / tacrolimus, and the bottom line corresponds to animals treated with vehicle + dexamethasone / tacrolimus.

[0029] [Figure 12]The technology provided can offer a reduction in hepatotoxicity. Cynomolgus monkeys were administered 5 × 10¹³ vg / kg of rAAV containing the nucleic acid sequence encoding the mCherry protein via a single intravenous (IV) injection in combination with an immunosuppressive regimen containing dexamethasone and tacrolimus (Dex / Tac) (N=3) or an immunosuppressive regimen containing prednisolone (Pred) (N=3). A control group of animals (N=2) received a vehicle control in combination with dexamethasone and tacrolimus. Dexamethasone, tacrolimus, and / or prednisolone were administered orally once daily. Animals were sacrificed on day 22, livers were collected, and lesions were examined. The treatment groups of animals are shown above. None of the listed liver lesions were observed in animals treated with the vehicle in combination with dexamethasone and tacrolimus. The size and fill of the circles represent the lesion severity score. For example, in animal 5001, apoptosis / single-cell necrosis was observed as mild, while hyperplasia in the bile ducts was observed as minimal. [Modes for carrying out the invention]

[0030] definition Where used herein, unless otherwise clearly indicated by the context, (i) the terms “a” or “an” may be understood to mean “at least one”; (ii) the terms “or” may be understood to mean “and / or”; (iii) the terms “comprising,” “comprise,” “including” (whether used with “not limited to”) and “include” (whether used with “not limited to”) may be understood to encompass the listed components or steps, whether presented by themselves or together with one or more additional components or steps; (iv) the term “another” may be understood to mean at least one additional / secondary; and (v) where a scope is provided, it includes endpoints.

[0031] Approximately: When the term “approximately” is used herein in reference to a value, it refers to a value that is similar in context to the referenced value. Generally, those skilled in the art will be familiar with the context and will understand the range of relevant degrees of variation that are encompassed by “approximately” in that context. For example, in some embodiments, the term “approximately” may encompass values ​​ranging from 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than 1% of the referenced value.

[0032] Adeno-associated virus (AAV): As used herein, the terms “adeno-associated virus” and “AAV” refer to all or part of viral particles of the Parvoviridae family and the Dependparvovirus genus. AAVs are small, replication-deficient, non-enveloped viruses. AAV may include, but is not limited to, AAV serotype 1, AAV serotype 2, AAV serotype 3 (including serotypes 3A and 3B), AAV serotype 4, AAV serotype 5, AAV serotype 6, AAV serotype 7, AAV serotype 8, AAV serotype 9, AAV serotype 10, AAV serotype 11, AAV serotype 12, AAV serotype 13, AAV serotype rh10, AAV serotype rh74, AAV from the HSC1-17 series, AAV from the CBr, CLv or CLg series, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, sheep AAV, goat AAV, shrimp AAV, and any variant of any of the aforementioned. AAV may also include engineered or chimeric versions of wild-type AAV, which include one or more insertions, deletions, and / or substitutions in the Cap polypeptide(s) affecting one or more characteristics of the wild-type AAV serotype, including, but not limited to, tropism and evasion of neutralizing antibodies (e.g., AAV-DJ, AAV-PHP.B, AAV-PHP.N, AAV.CAP-B1~AAV.CAP-B25 and their variants). Wild-type AAV is a replication defect and requires co-infection of cells with a helper virus (e.g., adenovirus, herpes, or vaccinia virus) or supplementation of helper virus genes to replicate.

[0033] Administration: As used herein, the term “administration” refers to the administration of the composition to a subject. Administration may be by any suitable route. For example, in some embodiments, administration may be intrabronchial (including by bronchial drip), buccal, enteral, intercutaneous, intraarterial, intradermal, gastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, subretinal, topical, intratracheal (including by intratracheal drip), percutaneous, vaginal, intravitreous, or any combination thereof. In some embodiments, administration may be subretinal. In some embodiments, preferred methods of administration reduce or prevent the immune response from the subject being treated.

[0034] Drugs: As used herein, the term “drugs” can mean any chemical class of compound or entity, including, for example, polypeptides, nucleic acids, sugars, lipids, small molecules, metals, or combinations thereof. As will be apparent from the context, in some embodiments, drugs are or include cells or organisms, or fractions, extracts, or components thereof. In some embodiments, drugs are or include natural products found in nature and / or obtained from nature. In some embodiments, drugs are or include one or more entities that are artificial in that they are designed, manipulated, and / or produced through the action of human hands, and / or not found in nature. In some embodiments, drugs may be available in isolated or pure forms, and in some embodiments, drugs may be available in crude forms. In some embodiments, potential drugs are provided as a collection or library that can be screened, for example, to identify or characterize the active ingredients among them. Some specific embodiments of the agents that may be used pursuant to this disclosure include small molecules, antibodies, antibody fragments, aptamers, siRNA, shRNA, miRNA, DNA / RNA hybrids, antisense oligonucleotides, ribozymes, peptides, peptide mimes, and small molecules. In some embodiments, the agent is a polymer or contains a polymer. In some embodiments, the agent is not a polymer and / or substantially does not contain any polymer. In some embodiments, the agent contains at least one polymeric moiety. In some embodiments, the agent lacks or substantially does not contain any polymeric moiety.

[0035] Comprising: A composition or method described herein as "comprising" or "including" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added to the scope of the composition or method. To avoid redundancy, any method described as "comprising" (or "comprises") one or more named elements or steps also describes a corresponding, more limited method "consisting essentially of" (or "consists essentially of") the same named elements or steps, meaning that the method includes the named essential elements or steps and may also include additional elements or steps that do not substantially affect the basic and novel features of the method. Furthermore, any method described herein as "comprising" or "consisting essentially of" one or more named elements or steps is also understood to describe a corresponding, more limited, and closed-end method that "consistes of" (or "consists of") any named element or step, in exchange for the exclusion of any other unnamed element or step. In any method disclosed herein, any known or disclosed equivalent of any named essential element or step may be substituted for that element or step.

[0036] Immunosuppressants: As used herein, the term “immunosuppressant” in its broadest sense refers to drugs, such as therapeutic agents, that suppress or reduce the activation, activity, or effectiveness of the immune system of a target. Exemplary immunosuppressants include abatacept, abrocitinib, adalimumab, alemtuzumab, anakinra, atacicept, azathioprine, baricitinib, basiliximab, belatacept, belimumab, bortezomib, certolizumab, clobarimab, cyclophosphamide, cyclosporine, daclizumab, dexamethasone, eculizumab, efalizumab, epratuzumab, etanercept, everolimus, fingolimod, fluorouracil, golimumab, hydroxychloroquine, and imurifi. This includes, but is not limited to, daze, infliximab, leflunomide, mercaptopurine, methotrexate, methylprednisolone, mycophenolate mofetil, mycophenolate sodium, ocrelizumab, ofatumumab, pimecrolimus, prednisone, prednisolone, ridafololimus, lilonacept, rituximab, ruxolitinib, secukinumab, sirolimus, tacrolimus, temsirolimus, tocilizumab, tofacitinib, upadacitinib, and bertuzumab. Additional immunosuppressants are known in the art.

[0037] Immunosuppressive regimen: As used herein, the term “immunosuppressive regimen” refers in its broadest sense to a therapeutic regimen comprising one or more immunosuppressants.

[0038] Nucleic Acids: As used herein, the term “nucleic acid” means, in its broadest sense, any compound and / or substance that is incorporated into or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is incorporated into or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As will be apparent from the context, in some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides), and in some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” is or contains RNA, and in some embodiments, “nucleic acid” is or contains DNA. In some embodiments, a nucleic acid is one or more native nucleic acid residues, or contains or consists of them. In some embodiments, a nucleic acid is one or more nucleic acid analogs, or contains or consists of them. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is one or more “peptide nucleic acids,” or contains or consists of them, which are known in the art, have peptide bonds instead of phosphodiester bonds in their backbone, and are considered to be within the scope of this disclosure. Alternatively, or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite bonds instead of phosphodiester bonds. In some embodiments, the nucleic acid is, contains, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In some embodiments, the nucleic acid is, includes, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, the nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to the native nucleic acid. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product such as RNA or protein. In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is prepared by one or more of the following: isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), regeneration in recombinant cells or systems, and chemosynthesis. In some embodiments, the nucleic acid comprises or can consist of one or more inhibitory nucleic acids (e.g., small RNA molecules). In some embodiments, the inhibitory nucleic acid comprises or consists of RNA molecules (e.g., small RNA molecules) that inhibit gene expression (e.g., via mRNA degradation) or inhibit translation (e.g., reducing the level of gene expression or the level of transcription translation compared to a relevant control). In some embodiments, the inhibitory nucleic acid comprises or consists of one or more siRNAs, miRNAs, shRNAs, gRNAs, or any combination thereof. In some embodiments, the inhibitory nucleic acid may be single-stranded or double-stranded.

[0039] Pharmaceutical Composition: As used herein, the term “pharmaceutical composition” refers to an activator formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the activator is present in a unit dose appropriate for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a population of interest. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including those adapted to: oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeting buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., sterile solutions or suspensions, or as sustained-release formulations; topical application, e.g., as creams, ointments, or controlled-release patches or sprays applied to the skin, lungs, or oral cavity; e.g., as pessaries, creams, or foams, e.g., transvaginal or rectal; sublingual; intraocular; transdermal; or transnasal, lung, and other mucosal surfaces. In some embodiments, the pharmaceutical composition is formulated for subretinal administration, for example, by subretinal injection.

[0040] Polypeptide: As used herein, the term "polypeptide" refers to any polymer chain of amino acids. In some embodiments, the polypeptide has a naturally occurring amino acid sequence. In some embodiments, the polypeptide has an amino acid sequence that does not exist naturally. In some embodiments, the polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through the action of human hands. In some embodiments, the polypeptide has an amino acid sequence that is encoded by a sequence that does not exist naturally (e.g., an engineered sequence that is designed and / or produced through the action of human hands to encode the polypeptide). In some embodiments, the polypeptide may contain or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, the polypeptide may contain or consist of only natural amino acids or only non-natural amino acids. In some embodiments, the polypeptide may contain D-amino acids, L-amino acids, or both. In some embodiments, the polypeptide may contain only D-amino acids. In some embodiments, the polypeptide may contain only L-amino acids. In some embodiments, the polypeptide may contain one or more pendant groups or other modifications, e.g., modifications or attachments to one or more amino acid side chains, at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, and the like, including combinations thereof. In some embodiments, the polypeptide may be cyclic and / or may contain a cyclic moiety. In some embodiments, the polypeptide may not be cyclic and / or may not contain any cyclic moiety. In some embodiments, the polypeptide may be linear. In some embodiments, the polypeptide may be or may contain a stapled polypeptide.In some embodiments, the term “polypeptide” may be affixed to the name of a reference polypeptide, activity, or structure, in which case it is used herein to refer to a polypeptide that shares a relevant activity or structure and can therefore be considered a member of the same class or family of polypeptides. For each such class, herein provides exemplary polypeptides within the class whose amino acid sequence and / or function is known, and / or those skilled in the art will recognize such exemplary polypeptides within the class whose amino acid sequence and / or function is known, and in some embodiments, such exemplary polypeptide is the reference polypeptide of the polypeptide class or family. In some embodiments, members of the polypeptide class or family exhibit significant sequence homology or identity with the reference polypeptide of the class, and in some embodiments, with all polypeptides within the class, share common sequence motifs (e.g., characteristic sequence elements), and / or share common activities (in some embodiments, at an equivalent level or within a specified range). For example, in some embodiments, the member polypeptide exhibits overall sequence homology or identity with the reference polypeptide, including at least about 30–40% and often exceeding or being more than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and / or often exhibiting very high sequence identity of 90%, or even more than 95%, 96%, 97%, 98%, or 99% (for example, in some embodiments, a conserved region that may be or may contain a characteristic sequence element). Such a conserved region typically comprises at least 3–4 amino acids, often up to 20 or more, and in some embodiments, the conserved region comprises at least one interval of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more consecutive amino acids. In some embodiments, the relevant polypeptide may include or consist of a fragment of the parent polypeptide.In some embodiments, a useful polypeptide may comprise or consist of multiple fragments, each of which is found within the same parent polypeptide in a spatial arrangement different from that found within the polypeptide of interest (for example, a fragment directly linked within the parent may be spatially separated within the polypeptide of interest, or vice versa, and / or the fragments may exist within the polypeptide of interest in a different order than within the parent), and thus the polypeptide of interest is a derivative of its parent polypeptide.

[0041] Subject: As used herein, the terms “subject” or “patient” refer to any organism to which the provided composition is administered or may be administered, for example, for experimental, diagnostic, preventive, cosmetic, and / or therapeutic purposes. In some embodiments, the subject is or includes cells or tissues. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is human. In some embodiments, the patient suffers from or is susceptible to one or more diseases, disorders, or conditions. In some embodiments, the patient exhibits one or more symptoms of a disease, disorder, or condition. In some embodiments, the patient is diagnosed with one or more diseases, disorders, or conditions.

[0042] Susceptible to disease: Individuals who are "suspended" to disease, disorder, and / or condition are individuals who have a higher risk of developing disease, disorder, and / or condition than the general population. In some embodiments, individuals susceptible to disease, disorder, and / or condition tend to have that disease, disorder, and / or condition. In some embodiments, individuals susceptible to disease, disorder, and / or condition do not need to be diagnosed with the disease, disorder, and / or condition. In some embodiments, individuals susceptible to disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, individuals susceptible to disease, disorder, and / or condition do not need to exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, individuals susceptible to disease, disorder, and / or condition develop the disease, disorder, and / or condition. In some embodiments, individuals susceptible to disease, disorder, and / or condition do not develop the disease, disorder, and / or condition.

[0043] Therapeutic Dose: As used herein, the term “therapeutic dose” means an amount sufficient to treat a disease, disorder, and / or condition when administered to a population that is suffering from or susceptible to the disease, disorder, and / or condition, according to a therapeutic drug regimen. In some embodiments, a therapeutic dose is one that reduces the incidence and / or severity of one or more symptoms of the disease, disorder, and / or condition, and / or delays their onset. Those skilled in the art will understand that the term “therapeutic dose” does not actually require that a successful treatment be achieved in a particular individual. Rather, a therapeutic dose may be an amount that, when administered to patients requiring such treatment, provides a particular desired pharmacological response in a significant number of subjects. It is specifically understood that certain subjects may actually be “refractory” to a “therapeutic dose.” For example, a refractory subject may have low bioavailability such that clinical efficacy is not obtained. In some embodiments, a reference to a therapeutically effective dose may refer to a quantity measured in one or more specific tissues (e.g., tissues affected by a disease, disorder, or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, or urine). Those skilled in the art will understand that in some embodiments, a therapeutically effective dose may be formulated and / or administered as a single dose. In some embodiments, a therapeutically effective dose may be formulated and / or administered in multiple doses, for example, as part of a drug regimen.

[0044] To treat: As used herein, the terms “treat,” “treatment,” or “treating” refer to any method used to partially or completely alleviate, improve, reduce, inhibit, prevent, delay the onset, reduce the severity, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. Treatment may be administered to subjects who show no signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to subjects who show only the initial signs of a disease, disorder, and / or condition, for example, to reduce the risk of developing a pathology associated with the disease, disorder, and / or condition.

[0045] Vectors: As used herein, the term “vector” refers to a nucleic acid molecule that can transport another nucleic acid to which it is ligated. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which an additional DNA segment can be ligated. Another type of vector is a viral vector, to which an additional DNA segment can be ligated into a viral genome. Certain vectors are capable of self-replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin for replication). Other vectors (e.g., non-episomal mammalian vectors) can be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Furthermore, certain vectors can direct the expression of a gene to which they are operably ligated. Such vectors are referred to herein as “expression vectors.” In some embodiments, the term “vector” refers to a drug capable of transporting nucleic acids, and the drug contains nucleic acids. In some embodiments, the vector contains or is a drug capable of transporting nucleic acids. [Modes for carrying out the invention]

[0046] rAAVs have become an increasingly important delivery method for gene therapy. However, the implementation of rAAVs is characterized by numerous challenges, including rAAV-related toxicity in specific organs, tissues, or cells. The use of rAAVs to target the central nervous system (CNS) is particularly characterized by reports of toxicity in the dorsal root ganglia, which may be partially attributable to the immune system response to one or more components of such rAAVs. Therefore, technologies, e.g., rAAVs, immunosuppressants, immunosuppressive regimens, and methods thereof, are necessary to address rAAV-related toxicity, including the toxicity reported in DRGs. In particular, this disclosure provides a variety of rAAVs, immunosuppressants, immunosuppressive regimens, and methods thereof. In some embodiments, the immunosuppressive regimen comprises one or more immunosuppressants, e.g., calcineurin inhibitors such as dexamethasone and / or macrolides (e.g., tacrolimus). In some embodiments, this disclosure provides methods comprising administering rAAVs and immunosuppressive regimens to a target. In some embodiments, rAAV, immunosuppressants, and / or immunosuppressive regimens are administered as described herein. In some embodiments, the techniques provided (e.g., immunosuppressants, immunosuppressive regimens, and methods thereof) can reduce dorsal root ganglion (DRG) toxicity, such as disease severity and / or incidence, and axonal degeneration.

[0047] Recombinant adeno-associated virus (rAAV) AAV is a small, non-enveloped virus that packages a single-stranded, linear DNA genome approximately 4.7–5 kb in length, according to reports. A member of the Parvoviridae family, AAV was discovered in 1965 as a contaminant of adenovirus isolates. Despite most humans (>70%) being seropositive for one or more serotypes, AAV has not been associated with any human or animal disease (Calcedo et al. (2011), Caldedo et al. (2009), each of which is incorporated herein by reference in its entirety). Both positive and negative DNA strands are equally well packaged, and infection can be initiated by particles containing either strand. The virus has a T=1 icosahedral capsid with a diameter of 25 nm, which is reportedly very stable. AAV has been demonstrated to be resistant to heat, acidic pH, and short-term exposure to proteases. The AAV genome contains three open reading frames (ORFs), rep (replica), cap (capsid), and aap (assembly activation protein) that collectively encode eight proteins (Rep78, Rep68, Rep52, Rep40, VP1, VP2, VP3, and AAP) expressed from three promoters (p5, p19, and p40). The mature capsid contains the amino acid sequence and packaged DNA of only one ORF (cap).

[0048] In some embodiments, the AAV is recombinant AAV (rAAV). rAAV vectors are widely used for inserting genes into mammalian cells (e.g., human cells). rAAV systems are generally well known in the art (see, for example, Kelleher and Vos, Biotechniques, 17(6):1110-17 (1994), Cotten et al., PNASUSA, 89(13):6094-98 (1992), Curiel, Nat Immun, 13(2-3):141-64 (1994), Muzyczka, Curr Top Microbiol Immunol, 158:97-129 (1992), and Asokan et al., Mol.Ther., 20(4):699-708 (2012), each of which is incorporated herein by reference in whole). Methods for generating and using rAAV vectors are described, for example, in U.S. Patent Nos. 5,139,941 and 4,797,368, each of which is incorporated herein by reference in whole.

[0049] In some embodiments, rAAV contains or is a naturally occurring AAV. In some embodiments, rAAV is a modified AAV or a variant of a naturally occurring AAV. In some embodiments, rAAV is generated by directed evolution, e.g., DNA shuffling, peptide insertion, or random mutagenesis, to introduce modifications to an AAV sequence that improve one or more properties for gene therapy. In some embodiments, such modifications evade or reduce immune responses or recognition by neutralizing antibodies and / or enable more efficient and / or targeted transduction (see, for example, Asuri et al., Molecular Therapy, 20.2:329-338 (2012), which is incorporated herein by reference in its entirety). In some embodiments, such modifications enable the identification of AAVs having modified tropisms. Methods using directed evolution to manipulate rAAVs can be found, for example, in U.S. Patent No. 8,632,764, which is incorporated herein by reference in its entirety. In some embodiments, modified AAVs are modified to contain specific tropisms.

[0050] In some embodiments, rAAV is U.S. Patent Nos. 7,906,111, 6,759,237, 7,105,345, 7,186,552, 9,163,260, 9,567,607, 4,797,368, 5,139,941, 5,252,479, 6,261,834, 7,718,424, 8,507,267, 8,846,389, 6,984,517, and No. 7,479,554, No. 6,156,303, No. 8,906,675, No. 7,198,951, No. 10,041,090, No. 9,790,472, No. 10,308,958, No. 10,526,61 No. 7, No. 7,282,199, No. 7,790,449, No. 8,962,332, No. 9,587,250, No. 10,590,435, No. 10,265,417, No. 10,485,883, No. 7,58 8,772, 8,067,01, 8,574,583, 8,906,387, 8,734,809, 9,284,357, 10,035,825, 8,628,966, 8, 927,514, 9,623,120, 9,777,291, 9,783,825, 9,803,218, 9,834,789, 9,839,696, 9,585,971, or These are derived from AAV genome sequences or variants described in U.S. Patent Publication No. 10,519,198, U.S. Patent Publication Nos. 2017 / 0166926, 2019 / 0015527, 2019 / 0054188, or 2020 / 0080109, or International Patent Application Nos. WO2018 / 160582, WO2020 / 028751, or WO2020 / 068990, each of which is incorporated herein by reference in whole.

[0051] In some embodiments, the rAAV is a dual or triple rAAV vector, for example, for the delivery of large payloads (e.g., payloads exceeding approximately 5 kb) and / or to address safety concerns associated with the administration of a single rAAV vector. In some embodiments, a dual rAAV vector comprises two separate rAAV vectors, each containing a fragment of the entire sequence of the large payload of interest, and when recombined, the fragments form the entire sequence or a functional portion of the large payload of interest. In some embodiments, a triple rAAV vector comprises three separate rAAV vectors, each containing a fragment of the sequence of the large payload of interest, and when recombined, the fragments form the entire sequence or a functional portion of the large payload of interest.

[0052] Multiple rAAV vectors (e.g., dual or triple rAAV vectors) can be delivered and co-transfected within the same cell, and fragments of the payload of interest are recombined to produce a single mRNA transcript of the entire payload of interest. In some embodiments, the fragmented payload contains non-duplicate sequences. In some embodiments, the fragmented payload contains specified duplicate sequences. In some embodiments, multiple rAAV vectors for dual or triple transfection may be of the same type (e.g., the same serotype and / or the same construct). In some embodiments, multiple rAAV vectors for dual or triple transfection may be of different types (e.g., different serotypes or different constructs).

[0053] In some embodiments, the rAAV vector comprises a single-stranded (ss) or self-complementary (sc) rAAV nucleic acid vector. In some embodiments, the rAAV vector comprises an expression construct and one or more regions containing an ITR sequence (e.g., a wild-type ITR sequence or an engineered ITR sequence) adjacent to the expression construct. In some embodiments, the rAAV vector is capsid-formed by a viral capsid. In some embodiments, the viral capsid comprises 60 capsid protein subunits. In some embodiments, the viral capsid comprises VP1, VP2, and VP3. In some embodiments, the VP1, VP2, and VP3 subunits are present in the capsid in a ratio of approximately 1:1:10, respectively.

[0054] AAV serotype The rAAV for use in the methods described herein may be any AAV serotype. AAV serotypes generally have different tropisms to target different tissues. In some embodiments, the rAAV serotype is selected based on tropism. Several AAV serotypes have been characterized, including, but not limited to, AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAVrh74, AAV-HSC1-17, AAV-CBr, AAV-CLv, AAV-CLg, AAV-DJ, AAV-PHP.B, AAV-PHP.N, AAV-PHP.eB, AAV2-retro, AAV9-retro, or AAV.CAP-B1~AAV.CAP-B25, as well as their variants or hybrids. In some embodiments, the rAAV serotype is a variant generated using directed evolution. A rAAV of a particular serotype may contain ITRs derived from a different serotype. For example, an rAAV may contain, or is, an AAV2 / 5, AAV2 / 6, AAV2 / 8, or AAV2 / 9 vector (e.g., an AAV6, AAV8, or AAV9 serotype with an AAV2 ITR).

[0055] In some embodiments, the AAV serotype has or contains a mutation in the wild-type AAV sequence. For example, the AAV serotype may have or contain a mutation in the AAV9 sequence (see, for example, Pulicherla et al., Molecular Therapy, 19(6):1070-1078 (2011), which is incorporated herein by reference in its entirety). rAAV serotypes (with corresponding nucleotide and amino acid substitutions) include AAV9.1 (G1594C;D532H), AAV6.2 (T1418A and T1436X;V473D and I479K), AAV9.3 (T1238A;F413Y), AAV9.4 (T1250C and A1617T;F417S), and AAV9.5 (A1235G, A1314T, A1642G, C1760T;Q412R, T548A, A587V). , AAV9.6(T1231A;F411I), AAV9.9(G1203A, G1785T;W595C), AAV9.10(A1500G, T1676C;M559T), AAV9.11(A1425T, A170 2C, A1769T; T568P, Q590L), AAV9.13 (A1369C, A1720T; N457H, T574S), AAV9.14 (T1340A, T1362C, T1560C, G1713A; L447 H), AAV9.16(A1775T;Q592L), AAV9.24(T1507C, T1521G;W503R), AAV9.26(A1337G, A1769C;Y446C, Q590P), AAV9.33(A 1667C;D556A), AAV9.34(A1534G, C1794T;N512D), AAV9.35(A1289T, T1450A, C1494T, A1515T, C1794A, G1816A;Q430L, Y484N, N98K, V606I), AAV9.40(A1694T, E565V), AAV9.41(A1348T, T1362C; T450S), AAV9.44(A1684C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T; N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G481R, W509R, L517V), 9.47 (G1241A, G1358A, A1669G, C1745T; S414N, G453D, K557E, T582I), AAV9.48 (C1445T, A1736T; P482L, Q579L), A AV9.50 (A1638T, C1683T, T1805A; Q546H, L602H), AAV9.53 (G1301A, A1405C, C1664T, G1811T; R134Q, S469R, A55 5V, G604V), AAV9.54 (CI531A, T1609A; L511I, L537M), AAV9.55 (T1605A; F535L), AAV9.58 (C1475T, C1579A; T49 2I, H527N), AAV.59(T1336C;Y446H), AAV9.61(A1493T;N498I), AAV9.64(C1531A, A1617T;L511I), AAV9.65(C13 35T, T1530C, C1568A;A523D), AAV9.68(C1510A;P504T), AAV9.80(G1441A, ;G481R)AAV9.83(C1402A, A1500T;P 468T, E500D), AAV9.87(T1464C, T1468C; S490P), AAV9.90(A1196T; Y399F), AAV9.91(T1316G, A1583T, C1782G, T AAV9 variants may include, but are not limited to, AAV9.93 (A1273G, A1421G, A1638C, C1712T, G1732A, A1744T, A1832T;S425G, Q474R, Q546H, P571L, G578R, T582S, D611V), AAV9.94 (A1675T;M559L), and AAV9.95 (T1605A;F535L). In certain embodiments, AAV9 variants include or are AAVhu68 or its variants (for example, described in International Publication WO2018 / 160585, which is incorporated herein by reference in its entirety). Other rAAVs are described, for example, in Sharma et al., Brain Res Bull., 81(2-3):273 (2010), which is incorporated herein by reference in its entirety.

[0056] AAV serotypes may be derived from any number of species. For example, rAAV may be, or contain, the avian AAV (AAAV) described in, for example, U.S. Patent No. 9,238,800, which is incorporated herein by reference in its entirety. rAAV serotypes may be, or contain, the bovine AAV (BAAV) described in, for example, U.S. Patent No. 9,193,769 or U.S. Patent No. 7,427,396, which are each incorporated herein by reference in their entirety. rAAV may be, or contain, the goat AAV described in, for example, U.S. Patent No. 7,427,396, which is incorporated herein by reference in its entirety. rAAV serotypes may also be any of the aforementioned variants or hybrids.

[0057] rAAVs include AAV9.68, AAV1, AAV10, AAV106.1 / hu.37, AAV11, AAV114.3 / hu.40, AAV12, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.1 / hu.43, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV16.12 / hu.11, AAV16.3, AAV16.8 / hu.10, AAV161.10 / hu.60, AAV161.6 / hu. u.61、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2、AAV2.5T、AAV2-15 / rh.62、AAV223.1、AAV223.2、AAV223.4、AAV223.5、AAV223.6、AAV223.7、AAV2-3 / rh.61、 AAV24.1、AAV2-4 / rh.50、AAV2-5 / rh.51、AAV27.3、AAV29.3 / bb.1、AAV29.5 / bb.2、AAV2G9、AAV-2-pre-miRNA-101、AAV3、AAV3.1 / hu.6、AAV3.1 / hu.9、AAV3. AV3-11 / rh.53、AAV3-3、AAV33.12 / hu.l7、AAV33.4 / hu.l5、AAV33.8 / hu.l6、AAV3-9 / rh.52、AAV3a、AAV3b、AAV4、AAV4-19 / rh.55、AAV42.12、AAV42-10、 AAV42-11、AAV42-12、AAV42-13、AAV42-15、AAV42-lb、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42-5a、AAV42-5b、AAV42-6b、AAV42-8、AAV42-aa、AA V43-1、AAV43-12、AAV43-20、AAV43-21、AAV43-23、AAV4f3-25、AAV43-5、AAV4-4、AAV44.1、AAV44.2、AAV44.5、AAV46.2 / hu.28、AAV46.6 / hu.29、AAV4-8 / r11.64、AAV4-8 / rh.64、AAV4-9 / rh.54、AAV5、AAV52.1 / hu.20、AAV52 / hu.19、AAV5-22 / rh.58、AAV5-3 / rh.57、AAV54.1 / hu.21、AAV54.2 / hu.22、AAV54.4R / hu.27、AAV54.5 / hu.23、AAV54.7 / hu.24、AAV58.2 / hu.25、AAV6、AAV6.1、AAV6.1.2、AAV6.2、AAV7、AAV7.2、AAV7.3 / hu.7、AAV8、AAV-8b、AAV-8h、AAV9、AAV9.11、AAV9.13、AAV9.16、AAV9.24、AAV9.45、AAV9.47、AAV9.61、AAV9.84、AAV9.9、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAV-b、AAVC1、AAVC2, AAVC5、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAV-DJ、AAV-DJ8、AAVF3 、AAVF5、AAV-h、AAVH-1 / hu.1、AAVH2、AAVH-5 / hu.3、AAVH6、AAVhE1.1、AAVhER1.14、AAVhEr1.16、AAVhEr1.18、AAVhER1.23、AAVhEr1.35、AAVhEr1.36、 AAVhEr1.5、AAVhEr1.7、AAVhEr1.8、AAVhEr2.16、AAVhEr2.29、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhEr2.4、AAVhEr3.1、AAVhu.1、AAVhu.10、AA Vhu.11、AAVhu.12、AAVhu.13、AAVhu.14 / 9、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.19、AAVhu.2、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23 .2、AAVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.3、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu. u.4、AAVhu.40、AAVhu.41、AAVhu.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu.48R3、AAVhu.49、AAVhu.5、AAVhu.51、AAVhu.52、AAVhu.53、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.6、 AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.7、AAVhu.8、AAVhu.9、AAVhu.t19、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVLG-9 / hu.39、AAV-LK01、AAV-LK02、AAVLK03、AAV-LK03、AAV-LK04、AAV-LK05、AAV-LK06、AAV-LK07、AAV-LK08、AAV-LK09、AAV-LK10、AAV-LK1 1、AAV-LK12、AAV-LK13、AAV-LK14、AAV-LK15、AAV-LK17、AAV-LK18、AAV-LK19、AAVN721-8 / rh.43、AAV-PAEC、AAV-PAEC11、AAV-PAEC12、AAV-PAEC2、AAV -PAEC4、AAV-PAEC6、AAV-PAEC7、AAV-PAEC8、AAVpi.1、AAVpi.2、AAVpi.3、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.18、 AAVrh.19、AAVrh.2、AAVrh.20、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.2R、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、 AAVrh.36、AAVrh.37、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.43、AAVrh.44、AAVrh.45、AAVrh.46、AAVrh.47、AAVrh.48、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.50、AAVrh.51、AAVrh.52、AAVrh.53、AAVrh.54、AAVrh.55、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.59, AAVrh.60, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.65, AAVrh.67, AAV rh.68, AAVrh.69, AAVrh.70, AAVrh.72, AAVrh.73, AAVrh.74, AAVrh.8, AAVrh.8R, AAVrh8R, AAVrh8R A586R variant, AAVrh8R R533A variant, BAAV, B P61 AAV, B P62 AAV, B P63 AAV, bovine AAV, goat AAV, Japanese AAV10, true type AAV(ttAAV), UPENN AAV10, AAV-LK16, AAAV, AAV Shuffle100-1, AAV Shuffle100-2, AAV This list may include, or be based on, any serotype selected from, or variants thereof, including, but not limited to, Shuffle100-3, AAV Shuffle100-7, AAV Shuffle10-2, AAV Shuffle10-6, AAV Shuffle10-8, AAV SM100-10, AAV SM100-3, AAV SM10-1, AAV SM10-2, and AAV SM10-8.

[0058] Capsid This disclosure encompasses the recognition that more than 110 different primate AAV capsid sequences have been reportedly isolated. Each of these AAV capsids, possessing a unique serological profile, is named as a specific AAV serotype. This disclosure further understands that at least 12 primate serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12) have been described. In some embodiments of this disclosure, capsids from any serotype may be used. In some embodiments, modified or manipulated capsids, including but not limited to those described herein, may be used in accordance with this disclosure.

[0059] This disclosure further encompasses the recognition that modifying or manipulating wild-type AAV capsids may be advantageous in achieving modified tissue tropism and / or immune system evasion. One way to achieve these advantages is to produce vectors in the presence of cap genes for multiple serotypes. Depending on the ratio of capsid proteins from each serotype, the resulting “mosaic” virions may exhibit complex tropism for a cell type, or, in some cases, acquire tropism not individually exhibited by any of the serotypes. Several studies have involved attaching exogenous molecules to the capsid. One example utilizes a bispecific antibody obtained by fusing the Fc regions of two different antibodies: an anti-capsid antibody and an anti-cell marker antibody, thereby conferring rAAV2 affinity to transduction-resistant megakaryocyte cell lines. Another example employs a method of biotinylating the capsid and then binding it to a streptavidin complex carrying epidermal growth factor or fibroblast growth factor. These methods were shown to increase the transduction of cells that highly express either epidermal growth factor or fibroblast growth factor receptor by at least 10 times.

[0060] This disclosure also recognizes that directly manipulating the cap gene for modification may be advantageous as an alternative to attaching molecules to the capsid surface. As one non-limiting example, green fluorescent protein (GFP) (238 amino acids) can be inserted into AAV2 VP1 and VP2. The transduction efficiencies of VP1-GFP and VP2-GFP vectors were 3 and 5 orders of magnitude lower than those of wild-type capsids, respectively, but transduction occurred in HeLa cells, suggesting tolerance to the inserted sequence in the capsid protein. As another non-limiting example, to modify the cap gene for tissue targeting, some researchers have inserted peptide sequences based on known ligand-receptor interactions or selected peptides from phage display libraries. Another strategy was to insert a random sequence of amino acids and then select the best-performing capsid in vitro. Instead of introducing target-specific peptides, some experiments have modified capsids in general until subsequent modifications directed towards a selected target. For example, the Fc portion of an antibody was inserted into the capsid, followed by the binding of different antibodies specific to receptors in various cell lines. Another such modification involves inserting a biotin-binding site into the capsid, thereby promoting metabolic biotinylation and enabling flexible targeting by any avidin-conjugated ligand. Several experiments have utilized peptide insertion as well as mosaic capsids containing both wild-type and modified capsid proteins, or combinations of multiple different modified capsid proteins. Other techniques are under investigation to circumvent the immune system, and these include coating the capsid with polymers.

[0061] Inverted terminal repeat (ITR) This disclosure recognizes that the AAV coding region is typically 145 nucleotides long in wild-type AAV and flanks a reverse terminal repeat (ITR) with a complex T-shaped structure. These repeats are the starting point for DNA replication and function as primary packaging signals (McLaughlin et al., 1988; Hauswirth et al., 1977, each of which is incorporated herein by reference in its entirety). This disclosure further recognizes that the ITR is the only cis-active sequence required to construct rAAV and is the only AAV coding sequence present in the AAV vector (McLaughlin et al. (1988); Samulski et al. (1989), each of which is incorporated herein by reference in its entirety). AAV ITRs have enhancer activity in the presence of Rep proteins, but have minimal promoter or enhancer activity in the absence of Rep proteins. Therefore, the transgene cloned into the AAV vector must be manipulated with appropriate enhancers, promoters, polyadenylation signals, and / or splice sites to ensure correct gene expression.

[0062] The rAAV ITR sequence may be derived from any rAAV serotype (e.g., AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAVrh74, AAV-HSC1-17, AAV-CBr, AAV-CLv, AAV-CLg, AAV-DJ, AAV-PHP.B, AAV-PHP.N, or AAV.CAP-B1~AAV.CAP-B25, as well as their variants or hybrids). In some embodiments, the ITRs of the Disclosure may include manipulated or modified ITRs using methods known in the Art. In some embodiments, the ITRs of the Disclosure may include one or more sequence modifications (e.g., deletions or substitutions) compared to the wild-type ITR sequence.

[0063] In some embodiments, the ITR sequence is, for example, incorporated in its entirety by reference in the following U.S. Patents: 7,906,111, 6,759,237, 7,105,345, 7,186,552, 9,163,260, 9,567,607, 4,797,368, 5,139,941, 5,252,479, 6,261,834, 7,718,424, 8,507,267, No. 8,846,389, No. 6,984,517, No. 7,479,554, No. 6,156,303, No. 8,906,675, No. 7,198,951, No. 10,041,090, No. 9,790,4 No. 72, No. 10,308,958, No. 10,526,617, No. 7,282,199, No. 7,790,449, No. 8,962,332, No. 9,587,250, No. 10,590,435, No. 10 ,265,417, 10,485,883, 7,588,772, 8,067,01, 8,574,583, 8,906,387, 8,734,809, 9,284,357, No. 10,035,825, No. 8,628,966, No. 8,927,514, No. 9,623,120, No. 9,777,291, No. 9,783,825, No. 9,803,218, No. 9,834,78 It is derived from one or more other serotypes described in serotype 9, 9,839,696, 9,585,971, or 10,519,198, U.S. Patent Publication 2017 / 0166926, 2019 / 0015527, 2019 / 0054188, or 2020 / 0080109, or one or more other serotypes described in International Patent Application WO2018 / 160582, WO2020 / 028751, or WO2020 / 068990.

[0064] ITR sequences and plasmids containing ITR sequences are known in the art and commercially available (for example, from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, CA; and Addgene, Cambridge, MA; as well as products and services described in Kessler et al., PNAS, 93(24):14082-7 (1996), Machida, Methods in Molecular Medicine, Viral Vectors for Gene Therapy Methods and Protocols. 10.1385 / 1-59259-304-6:201, Humana Press Inc. 2003. Chapter 10. Targeted Integration by Adeno-Associated Virus, and U.S. Patents No. 5,139,941 and No. 5,962,313, each of which is incorporated herein by reference in whole).

[0065] production Methods for producing and isolating rAAV using desired isolated nucleic acid sequences or vectors and capsids are well known in the art. rAAV can be produced and isolated according to any suitable method, e.g., methods described in Clement and Grieger, 2016, Grieger et al., 2016, and Martin et al., 2013, the contents of which are incorporated herein by reference in their entirety. While not wishing to be bound by any particular theory or process, a production method typically involves culturing a host cell containing a nucleic acid sequence or vector comprising a nucleic acid sequence encoding an AAV capsid protein or a fragment thereof (e.g., a cap gene), a functional rep gene, AAV ITRs (e.g., AAV5'ITR and AAV3'ITR), a nucleic acid sequence encoding a product of interest (e.g., a polypeptide, e.g., a wild-type polypeptide), and sufficient helper function to enable packaging of the recombinant AAV vector into the recombinant AAV capsid protein.

[0066] Components cultured in host cells to package isolated nucleic acid sequences or vectors within AAV capsids may be supplied to host cells in trans. Alternatively, any one or more required components (e.g., isolated nucleic acid sequences or vectors, rep sequences, cap sequences, and / or helper functions) may be supplied by stable host cells manipulated to contain one or more of the required components using methods known to those skilled in the art. Most preferably, such stable host cells contain one or more required components under the control of an inductive promoter. However, such one or more required components may also be under the control of a constitutive promoter. Examples of suitable promoters are provided herein. In yet another alternative, selected stable host cells may contain one or more selected components under the control of a constitutive promoter and one or more other selected components under the control of one or more inductive promoters. For example, stable host cells may be generated that originate from 293 cells (containing E1 helper function under the control of a constitutive promoter) but contain rep and / or cap proteins under the control of an inductive promoter. Furthermore, other stable host cells are known in the art or can be produced by those skilled in the art.

[0067] The isolated nucleic acid sequences or vectors, rep sequences, cap sequences, and helper functions necessary to produce the rAAVs of this disclosure can be delivered to a packaging host cell using any suitable genetic element (e.g., a vector). The selected genetic element can be delivered by any preferred method (e.g., transfection), including those described herein. Methods used to construct any embodiment of this disclosure are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for generating rAAV virions are well known, and the selection of preferred methods is not limited to this disclosure. See, for example, K. Fisher et al., 1993 and U.S. Patent No. 5,478,745.

[0068] In some embodiments, rAAV can be produced using a triple transfection method (for example, details relating to the triple transfection method are described in detail in U.S. Patent No. 6,001,650, which is incorporated herein by reference). Typically, such rAAV is produced by transfecting host cells with a suitable vector (containing a nucleic acid sequence encoding the product of interest, e.g., a polypeptide) which is packaged into rAAV particles, an AAV rep / cap vector, and a helper functional vector. The AAV rep / cap vector encodes rep and cap sequences that function in trans for productive AAV replication and capsid formation. In some embodiments, the AAV rep / cap vector facilitates efficient AAV vector production without producing any detectable wild-type AAV virions (e.g., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use with this disclosure include pHLP19 as described in U.S. Patent No. 6,001,650 and the pRep6cap6 vector as described in U.S. Patent No. 6,156,303, both of which are incorporated herein by reference in their entirety. Helper function vectors encode nucleotide sequences for non-AAV-derived viral and / or cellular functions (e.g., “helper functions”) on which AAV replication depends. Helper functions include, but are not limited to, functions required for AAV replication, and include parts involved in AAV gene transcription activation, stage-specific AAV mRNA splicing, AAV DNA replication, cap expression product synthesis, and AAV capsid assembly. Virus-based helper functions may be derived from any known helper viruses, such as adenoviruses, herpesviruses (other than herpes simplex virus type 1), and vaccinia viruses.

[0069] Additional methods for generating and isolating rAAV are described, for example, in U.S. Patent Nos. 7,790,449, 7,282,199, International Publication Nos. WO2003 / 042397, WO2005 / 033321, WO2006 / 110689, and 7,588,772, each of which is incorporated herein by reference in whole.

[0070] Immunosuppressants and regimens This disclosure provides, in particular, immunosuppressants and regimens for administration with rAAV. Various immunosuppressants are known in the art. Immunosuppressants may target various immune system components, including, for example, T cells, B cells, cytokines, or chemokines. In addition, immunosuppressants may target various molecular signaling pathways, including, for example, the calcineurin signaling pathway, the JAK / STAT signaling pathway, the mTOR signaling pathway, and / or the TNF signaling pathway. Some immunosuppressants target metabolic processes, including, for example, purine biosynthesis and / or pyrimidine biosynthesis. Immunosuppressants may include various structures, including, for example, antibodies, steroids, macrolides, purine analogs, or pyrimidine analogs. Any suitable immunosuppressant may be used in immunosuppressive regimens as described herein. Any suitable immunosuppressant may be used in the methods described herein.

[0071] In some embodiments, one or more immunosuppressants include alkylating agents. In some embodiments, one or more immunosuppressants include antimetabolites. In some embodiments, one or more immunosuppressants include B cell inhibitors. In some embodiments, one or more immunosuppressants include calcineurin inhibitors. In some embodiments, one or more immunosuppressants include complement inhibitors. In some embodiments, one or more immunosuppressants include cell proliferation inhibitors. In some embodiments, one or more immunosuppressants include interleukin-1 receptor antagonists. In some embodiments, one or more immunosuppressants include inosine-5'-monophosphate dehydrogenase (IMPDH) inhibitors. In some embodiments, one or more immunosuppressants include interleukin-1 receptor-associated kinase 4 (IRAK4) inhibitors. In some embodiments, one or more immunosuppressants include Janus kinase (JAK) inhibitors. In some embodiments, one or more immunosuppressants include mammalian target of rapamycin (mTOR) inhibitors. In some embodiments, one or more immunosuppressants include sphingosine-1-phosphate (S1P) receptor modulators. In some embodiments, one or more immunosuppressants include T cell inhibitors. In some embodiments, one or more immunosuppressants include tumor necrosis factor α (TNF-α) inhibitors. In some embodiments, one or more immunosuppressants include proteases, such as cysteine ​​proteases.

[0072] In some embodiments, one or more immunosuppressants include steroids. In some embodiments, one or more immunosuppressants include corticosteroids. In some embodiments, one or more immunosuppressants include glucocorticoids. In some embodiments, one or more immunosuppressants include dexamethasone. In some embodiments, one or more immunosuppressants include prednisone. In some embodiments, one or more immunosuppressants include prednisolone. In some embodiments, one or more immunosuppressants include methylprednisolone.

[0073] In some embodiments, one or more immunosuppressants include macrolides. In some embodiments, one or more immunosuppressants include tacrolimus. In some embodiments, one or more immunosuppressants include pimecrolimus. In some embodiments, one or more immunosuppressants include sirolimus (rapamycin). In some embodiments, one or more immunosuppressants include everolimus. In some embodiments, one or more immunosuppressants include temsirolimus. In some embodiments, one or more immunosuppressants include ridafololimus.

[0074] In some embodiments, one or more immunosuppressants include mycophenolic acid. In some embodiments, one or more immunosuppressants include mycophenolate salts. In some embodiments, one or more immunosuppressants include mycophenolate mofetil (MMF). In some embodiments, one or more immunosuppressants include mycophenolate sodium (MPS).

[0075] In some embodiments, one or more immunosuppressants include an antibody or its antigen-binding moiety. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody or its antigen-binding moiety is an anti-BDCA2 antibody or its antigen-binding moiety. In some embodiments, the antibody or its antigen-binding moiety is an anti-B lymphocyte-stimulating factor (BLyS) antibody or its antigen-binding moiety, e.g., belimumab. In some embodiments, the antibody or its antigen-binding moiety is an anti-C5 antibody or its antigen-binding moiety, e.g., clovalimab, eculizumab. In some embodiments, the antibody or its antigen-binding moiety is an anti-CD3 antibody or its antigen-binding moiety, e.g., muromonab-CD3. In some embodiments, the antibody is an anti-CD11a antibody or its antigen-binding moiety, e.g., efalizumab. In some embodiments, the antibody or its antigen-binding moiety is an anti-CD20 antibody or its antigen-binding moiety, e.g., ocrelizumab, ofatumumab, rituximab, bertuzumab. In some embodiments, the antibody or its antigen-binding portion is an anti-CD22 antibody or its antigen-binding portion, for example, epratuzumab. In some embodiments, the antibody or its antigen-binding portion is an anti-CD25 antibody or its antigen-binding portion, for example, basiliximab, daclizumab. In some embodiments, the antibody or its antigen-binding portion is an anti-CD40 antibody or its antigen-binding portion. In some embodiments, the antibody or its antigen-binding portion is an anti-CD40L antibody or its antigen-binding portion. In some embodiments, the antibody or its antigen-binding portion is an anti-CD52 antibody or its antigen-binding portion, for example, alemtuzumab. In some embodiments, the antibody or its antigen-binding portion is an anti-IL-1β antibody or its antigen-binding portion, for example, canakinumab. In some embodiments, the antibody or its antigen-binding portion is an anti-IL-2 antibody or its antigen-binding portion. In some embodiments, the antibody or its antigen-binding portion is an anti-IL-6R antibody or its antigen-binding portion, for example, tocilizumab.In some embodiments, the antibody or its antigen-binding moiety is an anti-IL-17A antibody or its antigen-binding moiety, for example, secukinumab. In some embodiments, the antibody or its antigen-binding moiety is an anti-TNF-α antibody or its antigen-binding moiety, for example, infliximab, adalimumab, golimumab, or certolizumab. In some embodiments, the antibody or its antigen-binding moiety is an anti-lymphocyte antibody or its antigen-binding moiety. In some embodiments, the antibody or its antigen-binding moiety is an anti-T cell antibody or its antigen-binding moiety. In some embodiments, the antibody is an anti-B cell antibody or its antigen-binding moiety.

[0076] In some embodiments, one or more immunosuppressants include anti-thymocyte globulin (ATG). In some embodiments, one or more immunosuppressants include anti-lymphocyte globulin (ALG). In some embodiments, one or more immunosuppressants include intravenous immunoglobulin (IVIG).

[0077] In some embodiments, one or more immunosuppressants include purine analogs. In some embodiments, one or more immunosuppressants include azathioprine (AZA). In some embodiments, one or more immunosuppressants include mercaptopurine (6-MP). In some embodiments, one or more immunosuppressants include pyrimidine analogs. In some embodiments, one or more immunosuppressants include fluorouracil (5-FU).

[0078] In some embodiments, one or more immunosuppressants include a fusion protein. In some embodiments, one or more immunosuppressants include the Fc region or a portion of IgG1 fused to the CTLA4 extracellular domain or a portion thereof. In some embodiments, one or more immunosuppressants include abatacept. In some embodiments, one or more immunosuppressants include beratacept. In some embodiments, one or more immunosuppressants include the Fc region or a portion of IgG1 fused to the binding domain or a portion of a transmembrane activator and CAML interactor (TACI). In some embodiments, one or more immunosuppressants include atacicept. In some embodiments, one or more immunosuppressants include the Fc region or a portion of IgG1 fused to a tumor necrosis factor (TNF) receptor, e.g., TNF receptor 2, or a portion thereof. In some embodiments, one or more immunosuppressants include etanercept. In some embodiments, one or more immunosuppressants include the IL-1RAcP extracellular domain or a portion thereof, and / or the IL-1R1 extracellular domain or a portion thereof fused to the Fc region or a portion of IgG1. In some embodiments, one or more immunosuppressants include lilonacept.

[0079] In some embodiments, one or more immunosuppressants include abrocitinib. In some embodiments, one or more immunosuppressants include anakinra. In some embodiments, one or more immunosuppressants include baricitinib. In some embodiments, one or more immunosuppressants include bortezomib. In some embodiments, one or more immunosuppressants include cyclophosphamide. In some embodiments, one or more immunosuppressants include cyclosporine. In some embodiments, one or more immunosuppressants include fingolimod. In some embodiments, one or more immunosuppressants include hydroxychloroquine. In some embodiments, one or more immunosuppressants include folic acid analogs. In some embodiments, one or more immunosuppressants include imirifidase. In some embodiments, one or more immunosuppressants include leflunomide. In some embodiments, one or more immunosuppressants include methotrexate. In some embodiments, one or more immunosuppressants include ruxolitinib. In some embodiments, one or more immunosuppressants include tofacitinib. In some embodiments, one or more immunosuppressants include upadacitinib.

[0080] In some embodiments, one or more immunosuppressants include any combination of any immunosuppressants described herein (e.g., one, two, three, four, five, six, seven, eight, nine, or more immunosuppressants described herein).

[0081] As described herein, one or more immunosuppressants may be administered to a subject. In some embodiments, two or more immunosuppressants may be administered to a subject. In some embodiments, three or more immunosuppressants may be administered to a subject. In some embodiments, four or more immunosuppressants may be administered to a subject. In some embodiments, five or more immunosuppressants may be administered to a subject.

[0082] As described herein, one or more immunosuppressants may be administered to a subject as part of an immunosuppressive regimen. In some embodiments, two or more immunosuppressants described herein are administered to a subject as part of an immunosuppressive regimen. In some embodiments, three or more immunosuppressants described herein are administered to a subject as part of an immunosuppressive regimen. In some embodiments, four or more immunosuppressants described herein are administered to a subject as part of an immunosuppressive regimen. In some embodiments, five or more immunosuppressants described herein are administered to a subject as part of an immunosuppressive regimen. In some embodiments, an immunosuppressive regimen described herein is administered to a subject.

[0083] In some embodiments, the immunosuppressive regimen comprises one or more immunosuppressants as described herein. In some embodiments, the immunosuppressive regimen comprises two or more immunosuppressants as described herein. In some embodiments, the immunosuppressive regimen comprises three or more immunosuppressants as described herein. In some embodiments, the immunosuppressive regimen comprises four or more immunosuppressants as described herein. In some embodiments, the immunosuppressive regimen comprises five or more immunosuppressants as described herein. In some embodiments, the immunosuppressive regimen comprises a therapeutically effective amount of one or more immunosuppressants as described herein. In some embodiments, the immunosuppressive regimen comprises a therapeutically effective amount of two or more immunosuppressants as described herein. In some embodiments, the immunosuppressive regimen comprises a therapeutically effective amount of three or more immunosuppressants as described herein. In some embodiments, the immunosuppressive regimen comprises a therapeutically effective amount of four or more immunosuppressants as described herein. In some embodiments, the immunosuppressive regimen comprises a therapeutically effective amount of five or more immunosuppressants as described herein.

[0084] In some embodiments, the immunosuppressive regimen comprises dexamethasone. In some embodiments, the immunosuppressive regimen comprises dexamethasone and one or more additional immunosuppressants as described herein. In some embodiments, the immunosuppressive regimen comprises dexamethasone and a calcineurin inhibitor. In some embodiments, the immunosuppressive regimen comprises dexamethasone, a calcineurin inhibitor, and one or more additional immunosuppressants as described herein. In some embodiments, the immunosuppressive regimen comprises dexamethasone and a macrolide. In some embodiments, the immunosuppressive regimen comprises dexamethasone, a macrolide, and one or more additional immunosuppressants as described herein. In some embodiments, the immunosuppressive regimen comprises dexamethasone and tacrolimus. In some embodiments, the immunosuppressive regimen comprises dexamethasone, tacrolimus, and one or more additional immunosuppressants as described herein. In some embodiments, the immunosuppressive regimen comprises dexamethasone and tofacitinib. In some embodiments, the immunosuppressive regimen comprises dexamethasone, tofacitinib, and one or more additional immunosuppressants described herein. In some embodiments, the immunosuppressive regimen comprises dexamethasone, tacrolimus, and mycophenolate mofetil (MMF). In some embodiments, the immunosuppressive regimen comprises dexamethasone, tacrolimus, MMF, and one or more additional immunosuppressants described herein. In some embodiments, the immunosuppressive regimen comprises dexamethasone, tacrolimus, and tofacitinib. In some embodiments, the immunosuppressive regimen comprises dexamethasone, tacrolimus, tofacitinib, and one or more additional immunosuppressants described herein.

[0085] Treatment methods As will be understood by those skilled in the art, rAAVs, immunosuppressive regimens, and immunosuppressants are useful for a variety of purposes. In some embodiments, the techniques provided (e.g., rAAVs, immunosuppressive regimens, immunosuppressants, and methods thereof) are useful for administering to a subject and / or treating a control.

[0086] Administration of viral vectors, such as rAAV, has been reported to be associated with toxicity in various organs, tissues, and / or cells of the recipient. For example, rAAV administration has been reported to be associated with hepatotoxicity. In addition, administration of rAAV, particularly those targeting the central nervous system (CNS), has been associated with dorsal root ganglion (DRG) toxicity in various models, including mice, rats, pigs, and non-human primates. For example, Bolt et al.,J Toxicol Sci.2021;46(2):57-68, Fader et al.,Mol Ther Methods Clin Dev.2022 Jun 9;25:264-277, Hordeaux et al.,Hum Gene Ther.2020 Aug;31(15-16):808-818, Hordeaux et al.,Mol Ther Methods Clin Dev.2018 Jul 14;10:68-78, Hinderer et al.,Hum Gene Ther.2018 Mar;29(3):285-298, Palazzi et al.,Hum Gene Ther.2022 Feb;33(3-4):175-187, Tukov et al.,Hum Gene Ther.2022 See Jul;33(13-14):740-756, each of which is incorporated herein by reference in its entirety. These reports describe the observation of lesions (e.g., axonal / nerve fiber degeneration, neuronal cell body degeneration / necrosis, increased immune cell infiltration, and / or gliosis) in the DRG and associated spinal cord segments and nerves after administration of rAAV. In particular, this toxicity has been observed with various rAAVs, including those with various different capsids and nucleic acids (e.g., various different promoters, various different transgenes). The causative factors of this DRG toxicity are not fully understood, but reports suggest that transgene overexpression may play a significant role. See, for example, Buss et al., Mol Ther Methods Clin Dev. 2022 Feb 1;24:342-354, which is incorporated herein by reference in its entirety.

[0087] Various approaches have been attempted to address the toxicity observed with the administration of viral vectors, such as rAAV. For example, rAAVs can be designed with various nucleic acid sequences (e.g., promoters, enhancers, or other 5'- or 3'-untranslated region elements) to alter transgene expression levels, and / or be administered via variable routes and dosages. However, such methodologies may not always be applicable or capable of reducing toxicity as desired. Attenuation of transgene expression in specific tissues (e.g., by inclusion of sequences recognized by specific miRNAs) has been attempted to reduce transgene expression in tissues susceptible to toxicity. See, for example, Hordeaux et al., Hum Gene Ther. 2020 Aug;31(15-16):808-818, which is incorporated herein by reference in its entirety. Other approaches involve implementing immunosuppressive regimens as an attempt to reduce or prevent potentially harmful immune responses to rAAV. For example, see Prasad et al., Hum Gene Ther. 2022 Dec;33(23-24):1228-1245, which is incorporated herein by reference in its entirety. However, the reduction of rAAV-mediated toxicity, particularly DRG toxicity, has not yet been effectively addressed. This disclosure identifies, among other things, potential causative factors in DRG toxicity and immunosuppressive regimens, immunosuppressants, and methods thereof that may reduce DRG toxicity. In some embodiments, the techniques provided (e.g., rAAV, immunosuppressive regimens, immunosuppressants, and methods thereof) reduce toxicity in a subject, e.g., DRG toxicity. In some embodiments, the techniques provided reduce the severity and / or incidence of lesions in one or more DRGs. In some embodiments, the techniques provided reduce neurodegeneration in one or more DRGs. In some embodiments, the techniques provided reduce neuronal cell body degeneration and / or necrosis in one or more DRGs. In some embodiments, the techniques provided reduce nerve fiber degeneration in one or more DRGs. In some embodiments, the provided technology reduces axonal degeneration in one or more DRGs.In some embodiments, the provided technology reduces mononuclear cell infiltration in one or more DRGs. In some embodiments, the provided technology reduces the severity and / or incidence of lesions in spinal cord tissue. In some embodiments, the provided technology reduces neurodegeneration in spinal cord tissue. In some embodiments, the provided technology reduces neuronal cell body degeneration and / or necrosis in spinal cord tissue. In some embodiments, the provided technology reduces nerve fiber degeneration in spinal cord tissue. In some embodiments, the provided technology reduces axonal degeneration in spinal cord tissue. In some embodiments, the provided technology reduces mononuclear cell infiltration in spinal cord tissue.

[0088] In some embodiments, the Disclosure provides a method for administering to a subject rAAV (e.g., a therapeutically effective dose of rAAV) and an immunosuppressive regimen provided herein. In some embodiments, the Disclosure provides a method for treating a subject, comprising administering to such subject rAAV and an immunosuppressive regimen provided herein.

[0089] In some embodiments, the Disclosure provides a method for reducing DRG toxicity in a subject, comprising administering rAAV and immunosuppressive regimens provided herein to such subject. In some embodiments, the Disclosure provides a method for reducing the severity and / or incidence of lesions in one or more DRGs in a subject, comprising administering rAAV and immunosuppressive regimens provided herein to such subject. In some embodiments, the Disclosure provides a method for reducing neurodegeneration in one or more DRGs in a subject, comprising administering rAAV and immunosuppressive regimens provided herein to such subject. In some embodiments, the Disclosure provides a method for reducing degeneration and / or necrosis of neuronal cell bodies in one or more DRGs in a subject, comprising administering rAAV and immunosuppressive regimens provided herein to such subject. In some embodiments, the Disclosure provides a method for reducing neurofibrous degeneration in one or more DRGs in a subject, comprising administering rAAV and immunosuppressive regimens provided herein to such subject. In some embodiments, the Disclosure provides a method for reducing axonal degeneration in one or more DRGs in a subject, comprising administering rAAV and immunosuppressive regimens provided herein to such subject. In some embodiments, the Disclosure provides a method for reducing mononuclear cell infiltration in one or more DRGs in a subject, comprising administering rAAV and immunosuppressive regimens provided herein to such subject.

[0090] In some embodiments, the Disclosure provides a method for reducing spinal toxicity in a subject, comprising administering rAAV and immunosuppressive regimens provided herein to such subject. In some embodiments, the Disclosure provides a method for reducing the severity and / or incidence of lesions in spinal tissue in a subject, comprising administering rAAV and immunosuppressive regimens provided herein to such subject. In some embodiments, the Disclosure provides a method for reducing neurodegeneration in spinal tissue in a subject, comprising administering rAAV and immunosuppressive regimens provided herein to such subject. In some embodiments, the Disclosure provides a method for reducing neuronal cell body degeneration and / or necrosis in spinal tissue in a subject, comprising administering rAAV and immunosuppressive regimens provided herein to such subject. In some embodiments, the Disclosure provides a method for reducing nerve fiber degeneration in spinal tissue in a subject, comprising administering rAAV and immunosuppressive regimens provided herein to such subject. In some embodiments, the Disclosure provides a method for reducing axonal degeneration in spinal cord tissue in a subject, comprising administering rAAV and immunosuppressive regimens provided herein to such subject. In some embodiments, the Disclosure provides a method for reducing mononuclear cell infiltration in spinal cord tissue in a subject, comprising administering rAAV and immunosuppressive regimens provided herein to such subject.

[0091] In some embodiments, the Disclosure provides a method for treating a subject, comprising administering to such subject an immunosuppressive regimen comprising rAAV and one or more immunosuppressants (e.g., immunosuppressants as described herein). In some embodiments, the method for treating a subject comprises administering to such subject an immunosuppressive regimen comprising rAAV and dexamethasone and one or more additional immunosuppressants (e.g., immunosuppressants as described herein). In some embodiments, the method for treating a subject comprises administering to such subject an immunosuppressive regimen comprising rAAV and dexamethasone and a calcineurin inhibitor. In some embodiments, the method for treating a subject comprises administering to such subject an immunosuppressive regimen comprising rAAV and dexamethasone, a calcineurin inhibitor, and one or more additional immunosuppressants (e.g., immunosuppressants as described herein). In some embodiments, the method for treating a subject comprises administering to such subject an immunosuppressive regimen comprising rAAV and dexamethasone, a calcineurin inhibitor, and MMF. In some embodiments, a method of treating a subject includes administering rAAV and an immunosuppressive regimen comprising dexamethasone, a calcineurin inhibitor, and tofacitinib to such a subject.

[0092] In some embodiments, the Disclosure provides a method for reducing DRG toxicity in a subject receiving rAAV, comprising administering such rAAV and an immunosuppressive regimen (e.g., one or more immunosuppressive regimens described herein) to such subject. In some embodiments, a method for reducing DRG toxicity in a subject receiving rAAV comprises administering such rAAV and an immunosuppressive regimen comprising dexamethasone and one or more additional immunosuppressants to such subject. In some embodiments, a method for reducing DRG toxicity in a subject receiving rAAV comprises administering such rAAV and an immunosuppressive regimen comprising dexamethasone and a calcineurin inhibitor to such subject. In some embodiments, a method for reducing DRG toxicity in a subject receiving rAAV comprises administering such rAAV and an immunosuppressive regimen comprising dexamethasone, a calcineurin inhibitor and one or more additional immunosuppressants to such subject. In some embodiments, a method to reduce DRG toxicity in a subject receiving rAAV includes administering such rAAV and an immunosuppressive regimen comprising dexamethasone, a calcineurin inhibitor, and MMF to such subject.

[0093] In some embodiments, the Disclosure provides a method for reducing the severity and / or incidence of lesions in one or more DRGs in a subject receiving rAAV, comprising administering such rAAV and an immunosuppressive regimen (e.g., an immunosuppressive regimen as described herein) to such subject. In some embodiments, a method for reducing the severity and / or incidence of lesions in one or more DRGs in a subject receiving rAAV comprises administering such rAAV and an immunosuppressive regimen comprising dexamethasone and one or more additional immunosuppressants to such subject. In some embodiments, a method for reducing the severity and / or incidence of lesions in one or more DRGs in a subject receiving rAAV comprises administering such rAAV and an immunosuppressive regimen comprising dexamethasone and a calcineurin inhibitor to such subject. In some embodiments, a method to reduce the severity and / or incidence of lesions in one or more DRGs in a subject receiving rAAV includes administering such rAAV and an immunosuppressive regime comprising dexamethasone, a calcineurin inhibitor, and one or more additional immunosuppressants to such subject. In some embodiments, a method to reduce the severity and / or incidence of lesions in one or more DRGs in a subject receiving rAAV includes administering such rAAV and an immunosuppressive regime comprising dexamethasone, a calcineurin inhibitor, and MMF to such subject. In some embodiments, a method to reduce the severity and / or incidence of lesions in one or more DRGs in a subject receiving rAAV includes administering such rAAV and an immunosuppressive regime comprising dexamethasone, a calcineurin inhibitor, and tofacitinib to such subject.

[0094] In some embodiments, the Disclosure provides a method for reducing axonal degeneration in one or more DRGs in a subject receiving rAAV, comprising administering rAAV and an immunosuppressive regimen (e.g., an immunosuppressive regimen comprising one or more immunosuppressants as described herein) to such subject. In some embodiments, a method for reducing axonal degeneration in one or more DRGs in a subject receiving rAAV comprises administering rAAV and an immunosuppressive regimen comprising dexamethasone and one or more additional immunosuppressants to such subject. In some embodiments, a method for reducing axonal degeneration in one or more DRGs in a subject receiving rAAV comprises administering such rAAV and an immunosuppressive regimen comprising dexamethasone and a calcineurin inhibitor to such subject. In some embodiments, a method for reducing axonal degeneration in one or more DRGs in a subject receiving rAAV comprises administering such rAAV and an immunosuppressive regimen comprising dexamethasone, a calcineurin inhibitor and one or more additional immunosuppressants to such subject. In some embodiments, a method to reduce axonal degeneration in one or more DRGs in a subject receiving rAAV includes administering such rAAV and an immunosuppressive regime comprising dexamethasone, a calcineurin inhibitor, and an MMF to such subject.

[0095] In some embodiments, DRG toxicity is reduced compared to the reference state. In some embodiments, DRG toxicity is reduced compared to administration of rAAV without the immunosuppressive regimen described herein. In some embodiments, the severity and / or incidence of lesions in one or more DRGs is reduced compared to the reference state. In some embodiments, the severity and / or incidence of lesions in one or more DRGs is reduced compared to administration of rAAV without the immunosuppressive regimen described herein. In some embodiments, axonal degeneration in one or more DRGs is reduced compared to the reference state. In some embodiments, axonal degeneration in one or more DRGs is reduced compared to administration of rAAV without the immunosuppressive regimen described herein.

[0096] In some embodiments, the reference state includes administration of rAAV without an immunosuppressive regimen described herein. In some embodiments, the reference state includes administration of rAAV with an alternative immunosuppressive regimen lacking one or more immunosuppressants compared to an immunosuppressive regimen described herein.

[0097] In some embodiments, the subject is a mammal. In some embodiments, the subject is a mouse. In some embodiments, the subject is a rat. In some embodiments, the subject is a dog. In some embodiments, the subject is a pig. In some embodiments, the subject is a non-human primate, such as a juvenile non-human primate. In some embodiments, the subject is a human. In some embodiments, the subject is an adult, such as a human adult. In some embodiments, the subject is a pediatric subject, such as a human child.

[0098] Administration and medication As described herein, compositions comprising rAAV and / or compositions comprising one or more immunosuppressants can be administered to a subject. In some embodiments, rAAV and / or one or more immunosuppressants are administered by preferred routes known in the art. In some embodiments, rAAV is administered intravenously, intrathecally, intraventricularly, intracisionally, intramuscularly, intraparenchymally, intracranially, intraocularly, intraarticularly, intranasally, intraosseously, intraalveolarly, intraarterially, intraperitoneally, orally, subcutaneously, sublingually, or submucosally. In some embodiments, one or more immunosuppressants are administered intravenously, intrathecally, intraventricularly, intracisionally, intramuscularly, intraparenchymally, intracranially, intraocularly, intraarticularly, intranasally, intraosseously, intraalveolarly, intraarterially, intraperitoneally, orally, subcutaneously, sublingually, or submucosally.

[0099] Various routes of administration for AAV (e.g., rAAV) are known in the art. In some embodiments, rAAV is administered systemically. In some embodiments, rAAV is administered intravenously, intrathecally, intraventricularly, intracisionally, intramuscularly, intraparenchymally, intracranially, intraocularly, intraarticularly, intranasally, intrathecally, and / or subcutaneously. In some embodiments, rAAV is administered intravenously. In some embodiments, rAAV is administered intrathecally. In some embodiments, rAAV is administered intraventricularly. In some embodiments, rAAV is administered intracisternally. In some embodiments, rAAV is administered intramuscularly. In some embodiments, rAAV is administered intraparenchymally. In some embodiments, rAAV is administered intracranially. In some embodiments, rAAV is administered intraocularly. In some embodiments, rAAV is administered intraarticularly. In some embodiments, rAAV is administered intranasally. In some embodiments, rAAV is administered intrathecally. In some embodiments, rAAV is administered subcutaneously.

[0100] In some embodiments, rAAV is about 10 7 ~about 10 18 It is administered in doses of the viral genome (vg). In some embodiments, rAAV is administered at approximately 10 7 vg, about 10 8 vg, about 109 vg, about 10 10 vg, about 10 11 vg, about 10 12 vg, about 10 13 vg, about 10 14 vg, about 10 15 vg, about 10 16 vg, about 10 17 vg, or about 10 18 is administered at a dose of vg.

[0101] In some embodiments, one or more immunosuppressive agents are administered intravenously, intrathecally, intraventricularly, intracisternally, intramuscularly, parenchymally, intracranially, intraocularly, intraosseously, intraarticularly, intranasally, subcutaneously, and / or orally. In some embodiments, one or more immunosuppressive agents are administered intravenously. In some embodiments, one or more immunosuppressive agents are administered intrathecally. In some embodiments, one or more immunosuppressive agents are administered intraventricularly. In some embodiments, one or more immunosuppressive agents are administered intracisternally. In some embodiments, one or more immunosuppressive agents are administered intramuscularly. In some embodiments, one or more immunosuppressive agents are administered parenchymally. In some embodiments, one or more immunosuppressive agents are administered intracranially. In some embodiments, one or more immunosuppressive agents are administered intraocularly. In some embodiments, one or more immunosuppressive agents are administered intraosseously. In some embodiments, one or more immunosuppressive agents are administered intraarticularly. In some embodiments, one or more immunosuppressive agents are administered intranasally. In some embodiments, one or more immunosuppressive agents are administered subcutaneously. In some embodiments, one or more immunosuppressive agents are administered orally.

[0102] In some embodiments, one or more immunosuppressants are administered every other day, once daily, twice daily, three times daily, or four times daily. In some embodiments, one or more immunosuppressants are administered every other day. In some embodiments, one or more immunosuppressants are administered once daily. In some embodiments, one or more immunosuppressants are administered twice daily. In some embodiments, one or more immunosuppressants are administered three times daily. In some embodiments, one or more immunosuppressants are administered four times daily.

[0103] In some embodiments, dexamethasone is administered intraosseously, intrathecally, intravenously, and / or orally. In some embodiments, dexamethasone is administered intraosseously. In some embodiments, dexamethasone is administered intrathecally. In some embodiments, dexamethasone is administered intravenously. In some embodiments, dexamethasone is administered orally.

[0104] In some embodiments, dexamethasone is administered every other day, once daily, twice daily, three times daily, or four times daily. In some embodiments, dexamethasone is administered every other day. In some embodiments, dexamethasone is administered once daily. In some embodiments, dexamethasone is administered twice daily. In some embodiments, dexamethasone is administered three times daily. In some embodiments, dexamethasone is administered four times daily.

[0105] In some embodiments, dexamethasone is administered in doses of approximately 0.01 mg / kg to approximately 10 mg / kg. In some embodiments, dexamethasone is administered in doses of approximately 0.1 mg / kg to approximately 10 mg / kg. In some embodiments, dexamethasone is administered in doses of approximately 0.1 mg / kg to approximately 5 mg / kg. In some embodiments, dexamethasone is administered in doses of approximately 0.1 mg / kg to approximately 2.5 mg / kg. In some embodiments, dexamethasone is administered in doses of approximately 0.25 mg / kg to approximately 2.5 mg / kg. In some embodiments, dexamethasone is administered in doses of approximately 0.1 mg / kg to approximately 1 mg / kg. In some embodiments, dexamethasone is administered in doses of approximately 0.5 mg / kg to approximately 1 mg / kg. In some embodiments, dexamethasone is administered in doses of approximately 0.1 mg / kg. In some embodiments, dexamethasone is administered at a dose of approximately 0.15 mg / kg. In some embodiments, dexamethasone is administered at a dose of approximately 0.2 mg / kg. In some embodiments, dexamethasone is administered at a dose of approximately 0.25 mg / kg. In some embodiments, dexamethasone is administered at a dose of approximately 0.3 mg / kg. In some embodiments, dexamethasone is administered at a dose of approximately 0.35 mg / kg. In some embodiments, dexamethasone is administered at a dose of approximately 0.4 mg / kg. In some embodiments, dexamethasone is administered at a dose of approximately 0.45 mg / kg. In some embodiments, dexamethasone is administered at a dose of approximately 0.5 mg / kg. In some embodiments, dexamethasone is administered at a dose of approximately 0.55 mg / kg. In some embodiments, dexamethasone is administered at a dose of approximately 0.6 mg / kg. In some embodiments, dexamethasone is administered at a dose of approximately 0.65 mg / kg. In some embodiments, dexamethasone is administered at a dose of approximately 0.7 mg / kg. In some embodiments, dexamethasone is administered at a dose of approximately 0.75 mg / kg. In some embodiments, dexamethasone is administered at a dose of approximately 0.8 mg / kg. In some embodiments, dexamethasone is administered at a dose of approximately 0.85 mg / kg.In some embodiments, dexamethasone is administered at a dose of approximately 0.9 mg / kg. In some embodiments, dexamethasone is administered at a dose of approximately 0.95 mg / kg. In some embodiments, dexamethasone is administered at a dose of approximately 1 mg / kg.

[0106] In some embodiments, dexamethasone is administered on each of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days prior to the administration of rAAV. In some embodiments, dexamethasone is administered on the same day as the administration of rAAV. In some embodiments, dexamethasone is administered on each day after the administration of rAAV for a period of approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer. In some embodiments, dexamethasone is administered every other day during the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days prior to the administration of rAAV. In some embodiments, dexamethasone is administered on the same day as the administration of rAAV. In some embodiments, dexamethasone is administered every other day after rAAV administration for approximately one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or longer.

[0107] In some embodiments, calcineurin inhibitors are administered intravenously and / or orally. In some embodiments, calcineurin inhibitors are administered intravenously. In some embodiments, calcineurin inhibitors are administered orally. In some embodiments, macrolides are administered intravenously and / or orally. In some embodiments, macrolides are administered intravenously. In some embodiments, macrolides are administered orally. In some embodiments, tacrolimus is administered intravenously and / or orally. In some embodiments, tacrolimus is administered intravenously. In some embodiments, tacrolimus is administered orally.

[0108] In some embodiments, the calcineurin inhibitor is administered every other day, once, twice, three, or four times a day. In some embodiments, the calcineurin inhibitor is administered every other day. In some embodiments, the calcineurin inhibitor is administered once a day. In some embodiments, the calcineurin inhibitor is administered twice a day. In some embodiments, the calcineurin inhibitor is administered three times a day. In some embodiments, the calcineurin inhibitor is administered four times a day. In some embodiments, the macrolide is administered every other day, once, twice, three, or four times a day. In some embodiments, the macrolide is administered every other day. In some embodiments, the macrolide is administered once a day. In some embodiments, the macrolide is administered twice a day. In some embodiments, the macrolide is administered three times a day. In some embodiments, the macrolide is administered four times a day. In some embodiments, tacrolimus is administered every other day, once daily, twice daily, three times daily, or four times daily. In some embodiments, tacrolimus is administered every other day. In some embodiments, tacrolimus is administered once daily. In some embodiments, tacrolimus is administered twice daily. In some embodiments, tacrolimus is administered three times daily. In some embodiments, tacrolimus is administered four times daily.

[0109] In some embodiments, the calcineurin inhibitor is administered in doses of approximately 0.01 mg / kg to approximately 10 mg / kg. In some embodiments, the calcineurin inhibitor is administered in doses of approximately 0.1 mg / kg to approximately 10 mg / kg. In some embodiments, the calcineurin inhibitor is administered in doses of approximately 0.1 mg / kg to approximately 5 mg / kg. In some embodiments, the calcineurin inhibitor is administered in doses of approximately 0.1 mg / kg to approximately 2.5 mg / kg. In some embodiments, the calcineurin inhibitor is administered in doses of approximately 0.25 mg / kg to approximately 2.5 mg / kg. In some embodiments, the calcineurin inhibitor is administered in doses of approximately 0.1 mg / kg to approximately 1 mg / kg. In some embodiments, the calcineurin inhibitor is administered in doses of approximately 0.5 mg / kg to approximately 1 mg / kg. In some embodiments, the calcineurin inhibitor is administered in doses of approximately 0.1 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 0.15 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 0.2 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 0.25 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 0.3 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 0.35 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 0.4 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 0.45 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 0.5 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 0.55 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 0.6 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 0.65 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 0.7 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 0.75 mg / kg.In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 0.8 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 0.85 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 0.9 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 0.95 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 1 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 1.1 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 1.2 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 1.3 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 1.4 mg / kg. In some embodiments, the calcineurin inhibitor is administered at a dose of approximately 1.5 mg / kg.

[0110] In some embodiments, tacrolimus is administered in doses of approximately 0.01 mg / kg to approximately 10 mg / kg. In some embodiments, tacrolimus is administered in doses of approximately 0.1 mg / kg to approximately 10 mg / kg. In some embodiments, tacrolimus is administered in doses of approximately 0.1 mg / kg to approximately 5 mg / kg. In some embodiments, tacrolimus is administered in doses of approximately 0.1 mg / kg to approximately 2.5 mg / kg. In some embodiments, tacrolimus is administered in doses of approximately 0.25 mg / kg to approximately 2.5 mg / kg. In some embodiments, tacrolimus is administered in doses of approximately 0.1 mg / kg to approximately 1 mg / kg. In some embodiments, tacrolimus is administered in doses of approximately 0.5 mg / kg to approximately 1 mg / kg. In some embodiments, tacrolimus is administered in doses of approximately 0.1 mg / kg. In some embodiments, tacrolimus is administered in doses of approximately 0.15 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 0.2 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 0.25 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 0.3 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 0.35 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 0.4 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 0.45 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 0.5 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 0.55 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 0.6 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 0.65 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 0.7 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 0.75 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 0.8 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 0.85 mg / kg.In some embodiments, tacrolimus is administered at a dose of approximately 0.9 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 0.95 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 1 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 1.1 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 1.2 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 1.3 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 1.4 mg / kg. In some embodiments, tacrolimus is administered at a dose of approximately 1.5 mg / kg.

[0111] In some embodiments, the calcineurin inhibitor is administered on each of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days prior to the administration of rAAV. In some embodiments, the calcineurin inhibitor is administered on the same day as the administration of rAAV. In some embodiments, the calcineurin inhibitor is administered on each day after the administration of rAAV for a period of approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer. In some embodiments, the calcineurin inhibitor is administered every other day during the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days prior to the administration of rAAV. In some embodiments, the calcineurin inhibitor is administered on the same day as the administration of rAAV. In some embodiments, calcineurin inhibitors are administered every other day after rAAV administration for approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer. In some embodiments, macrolides are administered on each of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days prior to rAAV administration. In some embodiments, macrolides are administered on the same day as rAAV administration. In some embodiments, macrolides are administered on each day after rAAV administration for approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer. In some embodiments, macrolides are administered every other day during the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days prior to rAAV administration. In some embodiments, macrolides are administered on the same day as rAAV administration. In some embodiments, macrolides are administered every other day after rAAV administration for approximately one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or longer.

[0112] In some embodiments, tacrolimus is administered on each of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days prior to rAAV administration. In some embodiments, tacrolimus is administered on the same day as rAAV administration. In some embodiments, tacrolimus is administered on each day after rAAV administration for a period of approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer. In some embodiments, tacrolimus is administered every other day during the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days prior to rAAV administration. In some embodiments, tacrolimus is administered on the same day as rAAV administration. In some embodiments, tacrolimus is administered every other day after rAAV administration for approximately one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or longer.

[0113] In some embodiments, mycophenolate mofetil (MMF) is administered intravenously and / or orally. In some embodiments, MMF is administered intravenously. In some embodiments, MMF is administered orally.

[0114] In some embodiments, mycophenolate mofetil (MMF) is administered every other day, once daily, twice daily, three times daily, or four times daily. In some embodiments, MMF is administered every other day. In some embodiments, MMF is administered once daily. In some embodiments, MMF is administered twice daily. In some embodiments, MMF is administered three times daily. In some embodiments, MMF is administered four times daily.

[0115] In some embodiments, mycophenolate mofetil (MMF) is administered in doses of approximately 0.1 mg / kg to approximately 200 mg / kg. In some embodiments, MMF is administered in doses of approximately 1 mg / kg to approximately 100 mg / kg. In some embodiments, MMF is administered in doses of approximately 10 mg / kg to approximately 75 mg / kg. In some embodiments, MMF is administered in doses of approximately 5 mg / kg to approximately 50 mg / kg. In some embodiments, MMF is administered in doses of approximately 10 mg / kg to approximately 50 mg / kg. In some embodiments, MMF is administered in doses of approximately 25 mg / kg to approximately 50 mg / kg. In some embodiments, MMF is administered in doses of approximately 5 mg / kg. In some embodiments, MMF is administered in doses of approximately 10 mg / kg. In some embodiments, MMF is administered in doses of approximately 15 mg / kg. In some embodiments, MMF is administered in doses of approximately 20 mg / kg. In some embodiments, MMF is administered at a dose of approximately 25 mg / kg. In some embodiments, MMF is administered at a dose of approximately 30 mg / kg. In some embodiments, MMF is administered at a dose of approximately 35 mg / kg. In some embodiments, MMF is administered at a dose of approximately 40 mg / kg. In some embodiments, MMF is administered at a dose of approximately 45 mg / kg. In some embodiments, MMF is administered at a dose of approximately 50 mg / kg.

[0116] In some embodiments, MMF is administered in doses of approximately 0.1g to approximately 10g. In some embodiments, MMF is administered in doses of approximately 0.1g to approximately 5g. In some embodiments, MMF is administered in doses of approximately 0.5g to approximately 5g. In some embodiments, MMF is administered in doses of approximately 1g to approximately 5g. In some embodiments, MMF is administered in doses of approximately 0.1g to approximately 2.5g. In some embodiments, MMF is administered in doses of approximately 0.5g to approximately 2.5g. In some embodiments, MMF is administered in doses of approximately 1g to approximately 2.5g. In some embodiments, MMF is administered in doses of approximately 0.5g. In some embodiments, MMF is administered in doses of approximately 0.6g. In some embodiments, MMF is administered in doses of approximately 0.7g. In some embodiments, MMF is administered in doses of approximately 0.8g. In some embodiments, MMF is administered in a dose of approximately 0.9 g. In some embodiments, MMF is administered in a dose of approximately 1 g. In some embodiments, MMF is administered in a dose of approximately 1.1 g. In some embodiments, MMF is administered in a dose of approximately 1.2 g. In some embodiments, MMF is administered in a dose of approximately 1.3 g. In some embodiments, MMF is administered in a dose of approximately 1.4 g. In some embodiments, MMF is administered in a dose of approximately 1.5 g. In some embodiments, MMF is administered in a dose of approximately 1.6 g. In some embodiments, MMF is administered in a dose of approximately 1.7 g. In some embodiments, MMF is administered in a dose of approximately 1.8 g. In some embodiments, MMF is administered in a dose of approximately 1.9 g. In some embodiments, MMF is administered in a dose of approximately 2 g. In some embodiments, MMF is administered in a dose of approximately 2.1 g. In some embodiments, MMF is administered in a dose of approximately 2.2 g. In some embodiments, MMF is administered in a dose of approximately 2.3 g. In some embodiments, MMF is administered in a dose of approximately 2.4 g. In some embodiments, MMF is administered in a dose of approximately 2.5 g.

[0117] In some embodiments, MMF is administered on each of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days prior to rAAV administration. In some embodiments, MMF is administered on the same day as rAAV administration. In some embodiments, MMF is administered on each day after rAAV administration for approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer. In some embodiments, MMF is administered every other day during the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days prior to rAAV administration. In some embodiments, MMF is administered on the same day as rAAV administration. In some embodiments, MMF is administered every other day after rAAV administration for approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer.

[0118] In some embodiments, the JAK inhibitor is administered intravenously and / or orally. In some embodiments, the JAK inhibitor is administered intravenously. In some embodiments, the JAK inhibitor is administered orally. In some embodiments, tofacitinib is administered intravenously and / or orally. In some embodiments, tofacitinib is administered intravenously. In some embodiments, tofacitinib is administered orally.

[0119] In some embodiments, the JAK inhibitor is administered every other day, once, twice, three times, or four times a day. In some embodiments, the JAK inhibitor is administered every other day. In some embodiments, the JAK inhibitor is administered once a day. In some embodiments, the JAK inhibitor is administered twice a day. In some embodiments, the JAK inhibitor is administered three times a day. In some embodiments, the JAK inhibitor is administered four times a day. In some embodiments, tofacitinib is administered every other day, once, twice, three times, or four times a day. In some embodiments, tofacitinib is administered every other day. In some embodiments, tofacitinib is administered once a day. In some embodiments, tofacitinib is administered twice a day. In some embodiments, tofacitinib is administered three times a day. In some embodiments, tofacitinib is administered four times a day.

[0120] In some embodiments, tofacitinib is administered in doses of approximately 0.01 mg / kg to approximately 10 mg / kg. In some embodiments, tofacitinib is administered in doses of approximately 0.1 mg / kg to approximately 10 mg / kg. In some embodiments, tofacitinib is administered in doses of approximately 0.1 mg / kg to approximately 5 mg / kg. In some embodiments, tofacitinib is administered in doses of approximately 0.1 mg / kg to approximately 2.5 mg / kg. In some embodiments, tofacitinib is administered in doses of approximately 0.25 mg / kg to approximately 2.5 mg / kg. In some embodiments, tofacitinib is administered in doses of approximately 0.1 mg / kg to approximately 1 mg / kg. In some embodiments, tofacitinib is administered in doses of approximately 0.5 mg / kg to approximately 1 mg / kg. In some embodiments, tofacitinib is administered in doses of approximately 0.1 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 0.15 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 0.2 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 0.25 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 0.3 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 0.35 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 0.4 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 0.45 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 0.5 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 0.55 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 0.6 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 0.65 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 0.7 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 0.75 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 0.8 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 0.85 mg / kg.In some embodiments, tofacitinib is administered at a dose of approximately 0.9 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 0.95 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 1 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 1.1 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 1.2 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 1.3 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 1.4 mg / kg. In some embodiments, tofacitinib is administered at a dose of approximately 1.5 mg / kg.

[0121] In some embodiments, tofacitinib is administered in doses of approximately 0.1 mg to approximately 20 mg. In some embodiments, tofacitinib is administered in doses of approximately 0.5 mg to approximately 15 mg. In some embodiments, tofacitinib is administered in doses of approximately 1 mg to approximately 10 mg. In some embodiments, tofacitinib is administered in doses of approximately 1 mg to approximately 5 mg. In some embodiments, tofacitinib is administered in doses of approximately 2.5 mg to approximately 10 mg. In some embodiments, tofacitinib is administered in doses of approximately 2.5 mg to approximately 5 mg. In some embodiments, tofacitinib is administered in doses of approximately 5 mg to approximately 10 mg. In some embodiments, tofacitinib is administered in doses of approximately 1 mg. In some embodiments, tofacitinib is administered in doses of approximately 2 mg. In some embodiments, tofacitinib is administered in doses of approximately 3 mg. In some embodiments, tofacitinib is administered in a dose of approximately 4 mg. In some embodiments, tofacitinib is administered in a dose of approximately 5 mg. In some embodiments, tofacitinib is administered in a dose of approximately 6 mg. In some embodiments, tofacitinib is administered in a dose of approximately 7 mg. In some embodiments, tofacitinib is administered in a dose of approximately 8 mg. In some embodiments, tofacitinib is administered in a dose of approximately 9 mg. In some embodiments, tofacitinib is administered in a dose of approximately 10 mg.

[0122] In some embodiments, tofacitinib is administered on each of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days prior to rAAV administration. In some embodiments, tofacitinib is administered on the same day as rAAV administration. In some embodiments, tofacitinib is administered on each day after rAAV administration for approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer. In some embodiments, tofacitinib is administered every other day during the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days prior to rAAV administration. In some embodiments, tofacitinib is administered on the same day as rAAV administration. In some embodiments, tofacitinib is administered every other day after administration of rAAV for approximately one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or longer.

[0123] Pharmaceutical composition In general, compositions comprising one or more rAAVs and compositions comprising one or more immunosuppressants of this disclosure may be administered in any form, including tablets, powders, or liquids, formulated on pharmaceutically acceptable carriers or excipients, depending on the patient's condition. Additionally, inactive components well known in the art, such as binders, fillers, coatings, preservatives, colorants, flavorings, and other additives, may be optionally formulated together with one or more administered agents described herein, or may be completely excluded if there is a risk of adverse side effects to the patient, such as increased inflammation or inhibition of absorption of certain compounds.

[0124] In some embodiments, the Disclosure provides a pharmaceutical composition comprising rAAV and / or an immunosuppressant. In some embodiments, the pharmaceutical composition comprises one or more immunosuppressants. In some embodiments, the pharmaceutical composition comprises two or more immunosuppressants. In some embodiments, the pharmaceutical composition comprises three or more immunosuppressants. In some embodiments, for example, for therapeutic and clinical purposes, rAAV and / or an immunosuppressant is provided as a pharmaceutical composition.

[0125] In some embodiments, the pharmaceutical composition is suitable for administering or delivering rAAV and / or one or more immunosuppressants to an area or part of the body affected by a disease, disorder, or condition. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the rAAV and / or immunosuppressant provided herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the rAAV or immunosuppressant and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutically acceptable carrier is a buffer.

[0126] In some embodiments, the pharmaceutical composition is formulated for injection (e.g., intravenous, intrathecal, or subcutaneous injection), oral administration, buccal administration, inhalation, nasal administration, topical administration, ophthalmic administration, or ear drops. In some embodiments, the pharmaceutical composition is a tablet, pill, capsule, liquid, inhalant, nasal spray solution, suppository, suspension, gel, colloid, dispersion, suspension, solution, emulsion, ointment, lotion, eye drops, or ear drops. In some embodiments, the pharmaceutical composition is formulated for administration via routes as described herein. In some embodiments, the pharmaceutical composition is formulated for intravenous, intrathecal, intraventricular, intracisional, intramuscular, intraparenchymal, intracranial, intraocular, intraarticular, intranasal, intraosseous, alveolar, intraarterial, intraperitoneal, oral, subcutaneous, sublingual, and / or submucosal administration.

[0127] Characterization and evaluation In some embodiments, the properties and / or activity of the immunosuppressants, immunosuppressive regimens, rAAVs, and methods provided may be characterized and / or evaluated using various techniques available to those skilled in the art, e.g., biochemical assays, cell-based assays, animal models, or clinical trials. Certain useful techniques are described in the examples. Those skilled in the art reading this disclosure will readily understand that other techniques (e.g., in vitro models (e.g., cell lines) or animal models) for various diseases, disorders, or conditions may be designed and / or utilized in accordance with this disclosure to evaluate the techniques provided (e.g., immunosuppressants, immunosuppressive regimens, rAAVs, or methods). [Examples]

[0128] Example 1. Immune system responses may play a causal role in dorsal root ganglion toxicity. We conducted a time course study of lesions to identify molecular events that occur before and simultaneously with lesion formation. rAAV containing nucleic acid sequences encoding human transgene proteins was analyzed at 3.68 × 10⁶ 13The drug was administered via the intracisrural route to cynomolgus monkeys at a dose of vg / animal. The control group of cynomolgus monkeys received the vehicle only. Tissue was collected at terminal time points (days 5, 9, 15, and 29). Histopathology confirmed the presence of lesions in the dorsal root ganglia (DRG), including DRG neurodegeneration / necrosis, DRG mononuclear cell infiltration, and nerve fiber degeneration in the spinal cord's posterior columns, in animals treated with rAAV, starting on day 15 post-AAV administration, with increasing severity and incidence on day 29 post-AAV administration. However, as shown in Figures 1-5, immunohistochemistry and in-situ hybridization revealed that immune cell foci were present as early as day 5 (CD68+ macrophages, NCR1 / NKp46+ natural killer cells, CD20+ B cells, CD4+ T cells) or day 9 (CD8+ T cells) before lesion formation, and increased over time (days 15 and 29) in rAAV-treated animals compared to vehicles-only animals (day 29). Pre-neurodegenerative immune cell infiltration may suggest a causal role in the immune response in DRG toxicity. In-situ hybridization of transgene expression identified highly transduced DRG neurons that preferentially underwent neurodegeneration and / or necrosis, suggesting that transgene expression may contribute to cellular stress and / or immune cell targeting in highly transduced neurons.

[0129] Example 2. The provided technology can provide reduction of dorsal root ganglion, trigeminal ganglion, and motor neuron toxicity. Non-human primates (2-3 year old cynomolgus monkeys) were given rAAV containing a nucleic acid sequence encoding a human transgene protein, at a dose of 3 × 10⁶ on day 1. 13The drug was administered via a single intracisional cisterna magna (ICM) injection at a dose of vg / animal. One group of animals (n=3) received rAAV alone. Another group of animals (n=3) received rAAV and an immunosuppressive regimen including (i) dexamethasone (0.5 mg) via intrathecal injection on day 1, (ii) dexamethasone (0.5 mg / kg) orally once daily from two days prior to the study conclusion, (iii) tacrolimus (1 mg / kg) orally once daily from two days prior to the study conclusion, and (iv) mycophenolate mofetil (50 mg / kg) orally twice daily from two days prior to the study conclusion. The control group of animals (n=3) did not receive rAAV or an immunosuppressive regimen.

[0130] Four weeks after administration of rAAV, animals were sacrificed and tissue was collected for histopathological analysis. DRG tissue was evaluated for lesions including mononuclear cell infiltration and neuronal cell body degeneration. Spinal cord tissue was evaluated for lesions including posterior column axonal degeneration, ventral glioma, and motor neuron degeneration. Trigeminal ganglion tissue was evaluated for lesions including mononuclear cell infiltration and neuronal cell body degeneration. Lesions were scored for severity on a scale of 0 to 5, where 0 represented no lesion (no affected tissue), 1 represented minimal (<10% affected tissue), 2 represented mild (10-25% affected tissue), 3 represented moderate (25-50% affected tissue), 4 represented significant (50-95% affected tissue), and 5 represented severe (>95% affected tissue). As shown in Figure 6, all animals in the control group were found to have minimal or no lesions in all examined tissues, while two of the three animals that received rAAV alone showed moderate to mild lesions in both the DRG and spinal cord. In contrast, all animals that received rAAV and the immunosuppressive regimen showed minimal or no lesions across most tissues, including the DRG and spinal cord. Furthermore, animals that received rAAV alone showed a slight increase in total protein and albumin in cerebrospinal fluid (CSF) compared to the control group, while animals that received rAAV in combination with the immunosuppressive regimen did not show such an increase. These data suggest that this immunosuppressive regimen reduces DRG and spinal cord histopathology caused by AAV gene therapy, and therefore suggest a causal role of the immune response in this AAV-mediated toxicity.

[0131] Example 3. The provided technology can provide reduction of dorsal root ganglion, spinal cord, and sympathetic ganglion toxicity. Non-human primates (cynomolgus monkeys) were given rAAV containing a nucleic acid sequence encoding a human transgene protein (different from the one used in Example 1 or 2) at a dose of 3 × 10⁶ on day 1. 13The drug was administered via a single intracisional cisterna magna (ICM) injection at a dose of vg / animal. Animals were treated with an immunosuppressive regimen including (i) dexamethasone (0.5 mg / kg) orally once daily from 2 days prior to the study conclusion, (ii) tacrolimus (1 mg / kg) orally once daily from 2 days prior to the study conclusion, and (iii) mycophenolate mofetil (50 mg / kg) orally twice daily from 2 days prior to the study conclusion. At the study conclusion (day 43), DRG tissue was evaluated for lesions including mononuclear cell infiltration and neuronal cell body degeneration. Spinal cord tissue was evaluated for lesions including posterior column axonal degeneration, ventral keratomas, and motor neuron degeneration. Trigeminal ganglion tissue was evaluated for lesions including mononuclear cell infiltration and neuronal cell body degeneration. Lesions were scored for severity on a scale of 0 to 5, where 0 represented no lesions (no affected tissue), 1 represented minimal lesions (<10% affected tissue), 2 represented mild lesions (10-25% affected tissue), 3 represented moderate lesions (25-50% affected tissue), 4 represented significant lesions (50-95% affected tissue), and 5 represented severe lesions (>95% affected tissue). As shown in Figure 7, vehicle-treated animals had no lesions or minimal lesions in all tissues examined, but rAAV (3 × 10⁻¹⁰) was also shown. 13 Three of the three animals administered vg) showed mild to moderate lesions in the DRG and spinal cord. In contrast, all animals treated with rAAV and immunosuppressive regimens showed no lesions or minimal lesions in the DRG and spinal cord tissue. These data support the ability of immunosuppressive regimens to reduce the severity and incidence of DRG and spinal cord lesions after AAV gene therapy encoding a protein transgene.

[0132] Example 4. The provided technology can provide reduction of dorsal root ganglia, nerve roots, spinal cord, and peripheral neurotoxicity. In non-human primates (cynomolgus monkeys), 4 × 10¹⁶ doses were administered via a single intrathecal (IT) injection through lumbar puncture (LP). 13Animals were administered rAAV containing a nucleic acid sequence encoding miRNA at a dose of vg / animal on day 1. Animals were treated with an immunosuppressive regimen including: (i) dexamethasone (0.5 mg / kg) orally once daily from 2 days prior to study conclusion, and (ii) tacrolimus (1 mg / kg) orally once daily from 2 days prior to study conclusion, and / or tofacitinib (1 mg / kg) orally once daily from 2 days prior to study conclusion. At study conclusion (day 43), DRG tissue was evaluated for lesions including mononuclear cell infiltration and neuronal cell body degeneration. Spinal cord tissue was evaluated for lesions including posterior column axonal degeneration, ventral glioma, and motor neuron degeneration. Sciatic nerve was evaluated for lesions including nerve fiber degeneration and mononuclear cell infiltration. Trigeminal ganglion tissue was evaluated for lesions including mononuclear cell infiltration and neuronal cell body degeneration. Lesions were scored for severity on a scale of 0 to 5, where 0 represented no lesions (no affected tissue), 1 represented minimal lesions (<10% affected tissue), 2 represented mild lesions (10-25% affected tissue), 3 represented moderate lesions (25-50% affected tissue), 4 represented significant lesions (50-95% affected tissue), and 5 represented severe lesions (>95% affected tissue). As shown in Figure 8, all vehicle-treated animals showed no lesions or minimal lesions in all tissues examined, but 4 × 10⁶ 13 Of the three animals administered vg / animal rAAV, three showed mild to moderate lesions in both the DRG and spinal cord. In contrast, 4 × 10⁶ rAAV in combination with the "triple" (dexamethasone, tacrolimus, and tofacitinib) immunosuppressive regimen showed mild to moderate lesions. 13 In animals administered vg rAAV, the incidence of lesions was reduced in both the spinal cord and DRG in all three animals, and the severity was also reduced. In combination with an immunosuppressive regimen of dexamethasone and tacrolimus, or dexamethasone and tofacitinib, 4 × 10 13In animals administered vg / animal rAAV, the incidence and severity of lesions were reduced. In summary, these data support the causal role of the immune response in DRG and spinal cord lesion formation in response to AAV gene therapy encoding miRNA transgenes, and demonstrate that lesion incidence and severity can be reduced when rAAV is administered in combination with an immunosuppressive regimen.

[0133] Example 5. The technology provided can offer in vivo toxicity reduction. A non-human primate (cynomolgus macaque) contains a nucleic acid sequence that encodes the mCherry protein, with a count of 5 × 10⁶ 13 vg / kg of rAAV was administered via a single intravenous (IV) injection on day 0 in combination with either an immunosuppressive regimen containing dexamethasone and tacrolimus (Dex / Tac) (N=3) or an immunosuppressive regimen containing prednisolone (Pred) (N=3). The animal control group (N=2) received vehicle control in combination with an immunosuppressive regimen containing dexamethasone and tacrolimus. Dexamethasone was administered orally once daily at a dose of 0.5 mg / kg from day 2 to day 21. Tacrolimus was administered orally once daily at a dose of 1 mg / kg from day 2 to day 21. Prednisolone was administered orally once daily at a dose of 3 mg / kg from day 2 to day 21. Blood and plasma samples were collected from animals at various time points to assess peripheral immunosuppressive drug exposure, plasma nerve filament H levels, and liver enzyme (AST, ALT, ALP, GGT, LDH) and total bilirubin (TBIL) levels. As shown in Figure 9, the implemented immunosuppressive regimens (Dex / Tac or Pred) achieved the target range of peripheral drug exposure for each drug (Dex, Tac, or Pred), as examined on day 21. Animals were sacrificed at the end of the study (day 22), tissues were collected, and lesions were examined. Certain exemplary data are shown in Figures 10, 11, and 12 below, and in Tables 1 and 2.

[0134] Immunosuppressive regimens containing dexamethasone and tacrolimus were able to reduce the neurotoxicity of rAAV treatment. As shown in Figure 10A, animals administered with an immunosuppressive regimen containing Dex / Tac in combination with rAAV showed lower plasma NF-H levels compared to animals administered with an immunosuppressive regimen containing Pred in combination with rAAV. Furthermore, animals administered with an immunosuppressive regimen containing Dex / Tac in combination with rAAV showed comparable levels of plasma NF-H compared to animals administered with an immunosuppressive regimen containing Dex / Tac in combination with the vehicle alone. These results indicate that administration of an immunosuppressive regimen containing Dex / Tac can provide a reduction in neurotoxicity from rAAV treatment, and can provide a greater reduction compared to administration of an immunosuppressive regimen containing Pred. In addition, tissue examination from treated animals confirmed that the level of mCherry expression from rAAV was related to the level of plasma NF-H observed in animals administered with Pred. In contrast, the level of mCherry expression from rAAV was not related to the levels of plasma NF-H observed in animals administered Dex / Tac. Exemplary immunohistochemical images showing mCherry expression can be seen in Figure 10B. In some embodiments, immunosuppressive regimens provided herein, e.g., those comprising dexamethasone and tacrolimus, can provide reduced neurotoxicity in subjects when treated with rAAV compared to different immunosuppressive regimens, e.g., those not comprising dexamethasone and tacrolimus and / or those comprising prednisolone.

[0135] As shown in Figure 10C and Table 1 below, animals administered an immunosuppressive regimen containing Dex / Tac in combination with rAAV showed a reduction in the incidence and severity of histopathological lesions in several tissues, including nerve tissue, e.g., dorsal root ganglia (DRGs), and cardiac tissue. In various tissues examined, animals administered an immunosuppressive regimen containing Dex / Tac in combination with rAAV showed comparable incidence and / or severity of histopathological lesions compared to animals administered the same immunosuppression but in combination with vehicle alone. In some embodiments, immunosuppressive regimens provided herein, e.g., immunosuppressive regimens containing dexamethasone and tacrolimus, can provide a reduced incidence and / or severity of lesions in the DRG in subjects when treated with rAAV, compared to different immunosuppressive regimens, e.g., immunosuppressive regimens without dexamethasone and tacrolimus and / or immunosuppressive regimens containing prednisolone. In some embodiments, immunosuppressive regimens provided herein, such as those comprising dexamethasone and tacrolimus, can provide a reduced incidence and / or severity of lesions in cardiac tissue in a subject during treatment with rAAV, compared to different immunosuppressive regimens, such as those not comprising dexamethasone and tacrolimus and / or those comprising prednisolone. [Table 1-1] [Table 1-2]

[0136] The reduction of hepatotoxicity during rAAV treatment by the immunosuppressive regimens tested was investigated through measurement of liver enzyme levels and analysis of liver lesions at autopsy. As depicted in Figure 11 and Table 2 below, in animals administered immunosuppressive regimens containing Dex / Tac in combination with rAAV, the AAV-mediated increase in liver enzymes was reduced compared to animals administered immunosuppressive regimens containing Pred in combination with rAAV. Increases in aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyltransferase (GGT) levels were particularly reduced by the use of Dex / Tac compared to the use of Pred (see Figures 11A, 11B, 11C, and Table 2). In animals administered with Pred, the maximum multiplier change from baseline was greater in individual animals for all liver enzymes compared to animals administered with Dex / Tac (Table 2). Total bilirubin levels were also examined in all animals, as shown in Figure 11D and Table 2. Levels in animals administered with Dex / Tac were within the normal limits, while a significant increase was observed in animals administered with Pred. In some embodiments, immunosuppressive regimens provided herein, e.g., those comprising dexamethasone and tacrolimus, can provide a reduced increase in liver enzyme levels in subjects, for example, from baseline, when treated with rAAV, compared with different immunosuppressive regimens, e.g., those comprising dexamethasone and tacrolimus, and / or those comprising prednisolone.

[0137] As well as increased levels of liver enzymes and total bilirubin, liver lesions were reduced in animals treated with an immunosuppressive regimen containing Dex / Tac in combination with rAAV compared to animals treated with an immunosuppressive regimen containing Pred in combination with rAAV. The observed liver lesions and their severity in individual animals are shown in Figure 12. In animals treated with an immunosuppressive regimen containing Dex / Tac in combination with rAAV, both the incidence and severity of liver lesions were reduced compared to animals treated with an immunosuppressive regimen containing Pred in combination with rAAV. In some embodiments, immunosuppressive regimens provided herein, such as those containing dexamethasone and tacrolimus, can provide a reduced incidence and / or severity of lesions in liver tissue in subjects when treated with rAAV, compared to different immunosuppressive regimens, such as those not containing dexamethasone and tacrolimus and / or immunosuppressive regimens containing prednisolone. [Table 2]

[0138] Equal portions While various embodiments are described and illustrated herein, those skilled in the art will readily assume various other means and / or structures for performing the function and / or obtaining one or more of the results and / or advantages described herein, and each of such variations and / or modifications will be considered to include. More generally, those skilled in the art will mean that all parameters, dimensions, materials and configurations described herein are examples, and will readily understand that actual parameters, dimensions, materials and / or configurations may depend on the particular one or more applications in which the teachings of this disclosure are used. Those skilled in the art will be able to recognize or confirm many equivalents of the embodiments of this disclosure by means of routine experimentation alone. Thus, it should be understood that the embodiments described herein are presented merely as examples, and within the scope of the appended claims and their equivalents, the claimed technology may be practiced in ways other than those specifically described and claimed. In addition, any combination of two or more features, systems, articles, materials, kits and / or methods is included within the scope of this disclosure, provided that such features, systems, articles, materials, kits and / or methods are not mutually inconsistent.

Claims

1. A method for treating a target, wherein the method is (a) Administering recombinant adeno-associated virus vector (rAAV), (b) administering an immunosuppressive regimen, The aforementioned immunosuppressive regimen, (i) Dexamethasone, and (ii) The method comprising a calcineurin inhibitor.

2. The method according to claim 1, wherein the calcineurin inhibitor comprises a macrolide.

3. The method according to claim 1 or 2, wherein the calcineurin inhibitor comprises tacrolimus.

4. The method according to any one of the prior claims, wherein the rAAV comprises a nucleic acid sequence encoding a polypeptide.

5. The method according to any one of the prior claims, wherein the rAAV includes a nucleic acid sequence encoding an RNA molecule.

6. The method according to any one of the prior claims, wherein the rAAV is administered intravenously, intrathecally, intraventricularly, intracisionally, intramuscularly, intraparenchymally, intracranially, intraocularly, intraarticularly, intranasally, and / or subcutaneously.

7. The method according to claim 6, wherein the rAAV is administered intravenously.

8. The method according to any one of the prior claims, wherein the dexamethasone is administered orally, intrathecally, and / or intravenously.

9. The method according to claim 8, wherein the dexamethasone is administered orally.

10. The method according to any one of the prior claims, wherein the dexamethasone is administered in a dose of approximately 0.01 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 5 mg / kg, approximately 0.25 mg / kg to approximately 2.5 mg / kg, or approximately 0.5 mg / kg to approximately 1 mg / kg.

11. The method according to any one of the prior claims, wherein the dexamethasone is administered daily.

12. The aforementioned dexamethasone, (i) On each of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, or more days prior to the administration of the rAAV, (ii) on the same day as the administration of the rAAV, and / or (iii) The method according to any one of the prior claims, wherein the rAAV is administered on each day after administration for a period of approximately one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or longer.

13. The method according to any one of the prior claims, wherein the calcineurin inhibitor is administered orally and / or intravenously.

14. The method according to claim 13, wherein the calcineurin inhibitor is administered orally.

15. The method according to any one of the prior claims, wherein the calcineurin inhibitor is administered in a dose of approximately 0.01 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 5 mg / kg, approximately 0.25 mg / kg to approximately 2.5 mg / kg, or approximately 0.5 mg / kg to approximately 1 mg / kg.

16. The method according to any one of the prior claims, wherein the calcineurin inhibitor is administered daily.

17. The calcineurin inhibitor described above (i) On each of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, or more days prior to the administration of the rAAV, (ii) on the same day as the administration of the rAAV, and / or (iii) The method according to any one of the prior claims, wherein the rAAV is administered on each day after administration for a period of approximately one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or longer.

18. The method according to any one of the prior claims, wherein the subject is a human being.

19. The method according to claim 18, wherein the subject is an adult.

20. The method according to claim 18, wherein the subject is a child.

21. The method according to any one of the prior claims, wherein dorsal root ganglion (DRG) toxicity is reduced compared to the administration of rAAV without the immunosuppressive regimen.

22. The method according to any one of the prior claims, wherein the severity and / or incidence of lesions in one or more DRGs is reduced compared to the administration of rAAV without the immunosuppressive regimen.

23. The method according to any one of the prior claims, wherein nerve fiber degeneration in one or more DRGs is reduced compared to the administration of rAAV without the immunosuppressive regimen.

24. The method according to any one of the prior claims, wherein axonal degeneration in one or more DRGs is reduced compared to the administration of rAAV without the immunosuppressive regimen.

25. The method according to any one of the prior claims, wherein neuronal cell body degeneration and / or necrosis in one or more DRGs is reduced compared to administration of rAAV without the immunosuppressive regimen.

26. The method according to any one of the prior claims, wherein mononuclear cell infiltration in one or more DRGs is reduced compared to administration of rAAV without the immunosuppressive regimen.

27. The method according to any one of the prior claims, wherein the immunosuppressive regimen further comprises an inosine monophosphate dehydrogenase (IMPDH) inhibitor and / or a Janus kinase (JAK) inhibitor.

28. The method according to claim 27, wherein the IMPDH inhibitor comprises mycophenolate mofetil (MMF).

29. The method according to claim 28, wherein the MMF is administered orally or intravenously.

30. The method according to claim 28 or 29, wherein the MMF is administered in doses of approximately 0.1 mg / kg to approximately 200 mg / kg, approximately 1 mg / kg to approximately 100 mg / kg, approximately 10 mg / kg to approximately 75 mg / kg, or approximately 25 mg / kg to approximately 50 mg / kg.

31. The method according to any one of claims 28 to 30, wherein the MMF is administered daily or twice daily.

32. The aforementioned MMF is (i) On each of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, or more days prior to the administration of the rAAV, (ii) on the same day as the administration of the rAAV, and / or (iii) The method according to any one of claims 28 to 31, wherein the rAAV is administered on each day after administration for a period of approximately one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or longer.

33. The method according to claim 27, wherein the JAK inhibitor comprises tofacitinib.

34. The method according to claim 33, wherein the tofacitinib is administered orally or intravenously.

35. The method according to claim 33 or 34, wherein tofacitinib is administered in a dose of approximately 0.01 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 5 mg / kg, approximately 0.25 mg / kg to approximately 2.5 mg / kg, or approximately 0.5 mg / kg to approximately 1 mg / kg.

36. The method according to any one of claims 33 to 35, wherein the tofacitinib is administered daily.

37. The aforementioned tofacitinib, (i) On each of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, or more days prior to the administration of the rAAV, (ii) on the same day as the administration of the rAAV, and / or (iii) The method according to any one of claims 33 to 36, wherein the rAAV is administered on each day after administration for a period of approximately one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or longer.

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